Compounds for the treatment and prevention of infections

ABSTRACT

Provided herein are compounds of Formula (I) or (II): (I) (II) pharmaceutically acceptable salts, tautomers, prodrugs, and stereoisomers thereof, and pharmaceutical compositions thereof, wherein X, R N , R 1 , R 3 , R 4 , p, and m are as defined herein. Such compounds and compositions have been found useful in the treatment or prevention of viral infections, e.g., polyomaviral infections, and are further envisioned useful in treatment or prevention of other pathogenic conditions associated with endosomal trafficking. Methods of treating or preventing an infection by a pathogen secreting an AB 5  toxin is also contemplated.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent applications, U.S. Ser. No. 61/671,998, filed Jul.16, 2012 and U.S. Ser. No. 61/799,043, filed Mar. 15, 2013, each ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Members of the polyomavirus family, Polyomaviridae, infect mammals(rodents, bovines, primates/humans) and birds (fowl, psittacines) andcan severely affect various organs in these subjects. So far, ten humanpolyomaviruses have been described. Two of these, JC-polyomavirus (JCPyVor JCV) and BK-polyomavirus (BKPyV or BKV) are established pathogenswhich are known to cause severe disease in humans. For example, inimmunosuppressed subjects, lytic infection of oligodendrocytes by JCVresults in the fatal demyelinating disease, progressive multifocalleukoencephalopathy (PML). BKV infection in immunosuppressed subjectsresults in kidney necrosis and polyomavirus-induced neuropathy (PVN).Two other closely-related polyomaviruses, KI-polyomavirus (KIPyV or KIV;Karolinska Institute) and WU-polyomavirus (WUPyV or WUV; WashingtonUniversity), discovered almost simultaneously in 2007, have beenisolated from respiratory secretions and may be associated withrespiratory tract infection. See, e.g., Allander et al., J. of Virol.(2007) 81: 4130-6; Gaynor et al., PLoS Pathogens (2007) 3: e64. Merkelcell polyomavirus (MCPyV or MCV), discovered in 2008, has been found tobe integrated in a large proportion of Merkel cell carcinomas of theskin. See, e.g., Feng et al., Science (2008) 319: 1096-100. In 2010,three new skin infecting polyomaviruses, HPyV6, HPyV7, andtrichodysplasia spinulosa-associated polyomavirus (TSPyV or TSV), werediscovered. See, e.g., Schowalter et al., Cell Host Microbe (2010)7:509-515; Meijden et al., PLoS Pathogens (2010) 6:e1001024. Forexample, TSV was discovered in proliferative skin lesions(trichodysplasia spinulosa) seen in immunosuppressed patients. Meijdenin supra. In March 2011, a ninth polyoma virus HPyV9, related to amonkey lymphotropic virus (LPV), was cultured from the blood ofimmunosuppressed patients. See, e.g., Trusch et al., J. Gen. Virol.(2012) 93:698-705. Most recently, in 2012, a new polyoma virus wasreported isolated from the stool of a healthy child from Malawi. See,e.g., Siebrasse et al., J. Virol. “Identification of MW polyomavirus, anovel polyomavirus in human stool” 86:10321-10326.

Polyomaviruses, such as JCV and BKV, are a highly common source ofchildhood and young adult infection. A large majority of theseinfections appear to cause little or no symptoms and are probablylifelong persistent among almost all adults. Diseases caused bypolyomavirus infections are most common among subjects who becomeimmunosuppressed due to AIDS or old age, or after organ transplantation.Unfortunately, effective vaccines or antiviral therapies targeting theseviruses do not currently exist. Thus, there remains a need for therapiesto treat and prevent viral infections such as polyomavirus infections.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the imine2-(((5-methylthiophen-2-yl)methylene)amino)-N-phenylbenzamide (RETRO-2)and/or the corresponding cyclized product,2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one(Retro-2^(cycl)) are inhibitors of polyomavirus (JCV, BKV, and SV40)infectivity. RETRO-2 was initially described in Stechmann et al., Cell(2010) 141:231-242 as an AB₅ toxin inhibitor against ricin andShiga-like toxin infectivity, but its use as an inhibitor of againstviral infectivity, such as polyomavirus infectivity, was not at thattime appreciated.

RETRO-2 rapidly converts to Retro-2^(cycl) in solution, and it is likelythat the active compound against polyomaviral infectivity isRetro-2^(cycl). A preliminary structure activity relationship (SAR)study of Retro-2^(cycl) indicates replacing the methyl group at position5 of the thiophenyl ring with an ethyl group to provide BU62382A4 doesnot negatively impact potency against polyomavirus infectivity. It isenvisioned that varying substitution at the 5-position of the thiophenylring and/or substitution on one or both of the phenyl rings may lead toother potent inhibitors of polyomaviral infection. It is furtherenvisioned that such compounds may also exhibit good activity againstother conditions, e.g., other pathogenic conditions associated withendosomal trafficking or sorting.

Thus, in one aspect, provided is a method of treating a viral infection,the method comprising administering to a subject suffering from orlikely to suffer from a viral infection an effective amount of acompound of Formula (I) or (II):

or a mixture thereof, or pharmaceutically acceptable salt, tautomer,prodrug, or stereoisomer thereof; wherein

, X, R¹, R², R³, R⁴, p and m are as defined herein.

In certain embodiments, X is S.

In certain embodiments, R¹ is halo or substituted or unsubstituted C₁₋₆alkyl. In certain embodiments, R¹ is methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆).

In certain embodiments, R^(N) is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl.

In certain embodiments, R^(N) is a group of formula:

wherein:

L¹ is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted alkenylene, or substituted or unsubstituted alkynylene;

each instance of R² is independently halo, —NO₂, —CN, —SCN, —OR^(A2),—SR^(A2), —N(R^(A2))₂, —C(═O)R^(A2), —OC(═O)R^(A2), —SC(═O)R^(A2),—NR′²C(═O)R^(A2), —S(═O)₂R^(A2), substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl,

each instance of R^(A2) is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, substitutedor unsubstituted thiol, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom, or twoR^(A2) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring;

or two R² groups are joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring; and

n is 0, 1, 2, 3, 4 or 5.

In certain embodiments, each instance of R² is independently halo, —NO₂,—OR^(A2). In certain embodiments, L¹ is a bond. In certain embodiments,L¹ is substituted or unsubstituted C₁alkylene, substituted orunsubstituted C₁alkylene, substituted or unsubstituted C₁alkylene.

In certain embodiments, m is 0 or 1. In certain embodiments, eachinstance of R³ is independently halo or substituted or unsubstitutedalkyl.

In certain embodiments, R⁴ is hydrogen. In certain embodiments,

corresponds to a double bond.

In yet another aspect, provided is a method of treating a pathogeniccondition associated with endosomal trafficking, the method comprisingadministering to a subject suffering from or likely to suffer from thecondition an effective amount of a compound of Formula (I) or (II), or amixture thereof, or pharmaceutically acceptable salt, tautomer, prodrug,or stereoisomer thereof; herein

, X, R¹, R², R³, R⁴, p, and m are as defined herein. In certainembodiments, the pathogenic condition is a bacterial infection. Incertain embodiments, the pathogenic condition is a viral infection.

In certain embodiments, the compound inhibits retrograde endosomaltransport of the pathogen (viral) genome to the cell nucleus.

In certain embodiments, the viral infection is an infection caused byhuman papillomavirus (HPV). In certain embodiments, the HPV is HPV-16 orHPV-18.

In certain embodiments, the viral infection is an infection caused byhuman immunodeficiency virus (HIV). In certain embodiments, the HIV isHIV type 1 (HIV-1).

In certain embodiments, the viral infection is an infection caused byinfluenza virus.

In certain embodiments, the viral infection is an infection caused bypolyomavirus. In certain embodiments, the polyomavirus isJC-polyomavirus, BK-polyomavirus KI-polyomavirus, WU-polyomavirus,Merkel cell polyomavirus, HPyV6, HPyV7, trichodysplasiaspinulosa-associated polyomavirus, HPyV9, MW polyomavirus, or the monkeypolyomavirus SV40.

In still yet another aspect, provided is a method of treating aninfection by a pathogen secreting AB₅ toxin, the method comprisingadministering to a subject suffering from or likely to suffer from theinfection an effective amount of a compound of Formula (I) or (II). Incertain embodiments, the pathogen secreting AB₅ toxin is bacteria. Incertain embodiments, pathogen is E. coli. In certain embodiments, theAB₅ toxin is selected from the group consisting of ricin, Shiga toxin,Shiga-like toxins, cholera toxin, heat-labile enterotoxin, pertussistoxin, and subtilase cytotoxin.

In yet another aspect, provided are novel compounds of Formula (I) and(II), or pharmaceutically acceptable salts, tautomers, prodrugs, orstereoisomers thereof, provided the compound is not:

In still yet another aspect, provided are pharmaceutical compositionscomprising a compound of Formula (I) and (II), or pharmaceuticallyacceptable salts, tautomers, prodrugs, or stereoisomers thereof, and apharmaceutically acceptable excipient. In certain embodiments, thepharmaceutical composition is useful as a medicament for the treatmentand prevention of an infection or condition as described herein.

The details of one or more embodiments of the invention are set forth inthe Detailed Description of Certain Embodiments, as described below.Other features, objects, and advantages of the invention will beapparent from the Definitions, Examples, Figures, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict that Retro-2^(cycl) prevents infectivity of threepolyomaviruses. FIG. 1A: Dose dependent effect of Retro-2^(cycl)treatment on infection. Cells were pre-incubated with the indicatedconcentrations of Retro-2^(cycl) prior to infection with JCpyV, BKpyV,and SV40 at an MOI of 0.5. Cells were fixed and stained for VP1 andinfected cells were scored by flow cytometry. Infections were normalizedto a vehicle control treated infected sample. Data represents the meanof three replicates and error bars represent the standard deviation.FIG. 1B: Retro-2^(cycl) does not block infection of Adenoviruspseudovirus. Vero cells were pretreated with equivalent concentrationsof Retro-2^(cycl) as A prior to infection with an Ad5-GFP pseudovirus atan MOI of 0.5. Cells were detached at 72 hpi and GFP positive cells werescored by flow cytometry and normalized to a DMSO treated sample. Datarepresents the mean of three replicates and error bars indicate standarddeviation.

FIG. 2 depicts the viability of cells treated with Retro-2^(cycl).

FIG. 3 depicts the inhibitory activity of Retro-2^(cycl) against analready established infection of cells by JCV, BKV, and SV40polyomaviruses. Retro-2^(cycl) prevents spread of virus in a multicyclegrowth assay. Cells were infected with JCpyV, BKpyV, or SV40 at an MOIif 0.01 and allowed to replicate for 72 h. Cells were then treated with0.1 mM Retro-2^(cycl) and this culture media was replaced daily withfresh media containing 0.1 mM Retro-2^(cycl). Cells were scored forinfection every three days by flow cytometry. Data represents the meanof three replicates and error bars indicate standard deviation.

FIG. 4 depicts a reduction in viral loads of polyomavirus (JCV, BKV,SV40) infected cells treated with Retro-2^(cycl). Retro-2^(cycl) treatedcultures release less infectious virus into culture media. Tissueculture media from the infected cultures in FIG. 3 was harvested everythree days and used to infect nave cells that were not treated withRetro-2^(cycl). Data represents the mean of three replicates and errorbars indicate standard deviation.

FIGS. 5A-5B depicts inhibition of JCV induced cytopathic effects incells treated with Retro-2^(cycl) (FIG. 5A) or with the DMSO vehiclecontrol (FIG. 5B).

FIG. 6 depicts the time course of inhibition of JCV, BKV, and SV40infection in cells treated with Retro-2^(cycl).

FIG. 7 depicts the lack of inhibition of cell binding by polyomavirusesor cholera toxin B subunit in cells pretreated with Retro-2^(cycl).

FIGS. 8A-8E depict the structures and inhibitory activities of Retro-2analogs. FIG. 8A: Condensation of 2-aminobenzailide with4-methyl-2-thiophencarboxaldehyde in methanol yields two products termedRetro-2 and Retro-2^(cycl). These compounds were separated andindependently characterized. FIG. 8B: The structure of Retro-2^(cycl),shown in ball and stick representation, was solved by x-ray diffractionand was verified to be a dihydroquinazolanone. FIG. 8C: Retro-2^(cycl)protects cells from infection by polyomaviruses. Cells were pretreatedwith Retro-2 analogs and infected with JCPyV at an MOI of 0.5. Retro-2and Retro-2^(cycl) were shown to be equally effective in reducinginfectivity, suggesting that Retro-2 rapidly converts to Retro-2^(cycl)in solution. Conversely, a reduced form of Retro-2 or a meta substitutedform of Retro-2, which cannot cyclize, poorly inhibit JCPyV infectivityAs a positive control, cells were incubated with 20 ng/mL of brefeldinA, and this was shown to be highly neutralizing of JCPyV infectivity.Cells were fixed and stained for VP1 and infected cells were scored byflow cytometry. Infected cells were normalized to a vehicle treatedcontrol and an uninfected, vehicle control sample was included to ensureno background signal existed. Data represent the average of triplicatesamples. Error bars indicate standard deviation. FIG. 8D: Treatment ofRetro-2 with sodium cyanoborohydride and acetic acid in methanol resultsin the formation of Retro-2^(red), which is unable to cyclize.Observation of such a reaction indicates that Retro-2 and Retro-2^(cycl)are in equilibrium under protic conditions. FIG. 8E: Retro-2^(meta), aregioisomer of Retro-2 that is also unable to cyclize.

FIG. 9 depicts endocytosis of polyomiruses is not inhibited by treatmentwith Retro-2^(cycl). Cells were chilled to prevent endocytosis, andAlexa fluor 488 labeled J^(CpyV), BKpyV, or SV40 was bound to cells for1 h on ice. Unbound virus was removed by extensive washing and sampleswere heated for 120 min prior to fixation. Cells were imaged by confocalmicroscopy and images were captured before (green, labeled a) and after(red, labeled b) trypan blue addition to samples. Trypan blue willquench the fluorescence of solvent accessible virions, resulting ininternalized virions being pseudocolored yellow (labeled c).

FIGS. 10A-10C depict Retro-2^(cycl) inhibits ER trafficking ofpolyomaviruses. FIG. 10A: Colocalization was assessed using Manderscoefficients of colocalization and at least five cells were analyzed persample. Error bars denote standard deviation. FIG. 10B: Cells werepretreated with the indicated drug for 0.5 h prior to inoculation withJCpyV, BKpyV, or SV40 at an MOI of 1 or CTxB (2.0 μg/mL). Cells werethen incubated for 8 h with the indicated drug and fixed andpermeabilized. Cells were then stained with a polyclonal antibody to PDIto stain the ER (red) or a monoclonal antibody to SV40 (green). Arrowsindicate colocalization between virions and PDI. FIG. 10C:Retro-2^(cycl) treatment redistributed JCPyV to early endosomes. Cellswere transfected with early endosomal and ER markers (Early endosome(Rab5-RFP) in green; Endoplasmic reticulum (CFP-HO) in blue; JCV inred). In cells treated with Retro-2^(cycl), more virions are seen tocolocalize with Rab5 positive enodosomes and less colocalization is seenwith the ER.

FIG. 11A-11C depict Retro-2^(cycl) inhibits VP2 exposure ofpolyomaviruses. FIG. 11A: VP2 is exposed at time points late duringinfection. Cells were pretreated with the indicated drug and theninoculated with JCpyV, BKpyV, or SV40 at an MOI of 10 in the presence ofdrug prior to incubation for 10 h before fixation and staining for VP2.VP2 punctae and nuceli are indicated with arrows (punctae are also shownin green and nuclei are show in blue). FIG. 11B: VP2 is exposed in theER. Cells were incubated with JCpyV for 10 h, then fixed and stained forVP1 (green), VP2 (red), PDI (purple), and the nucleus was stained withBOBO-3 (blue). Enlarge portions of the fluorescence micrograph is shownon the right with panels for individual antibody staining FIG. 11C:Quantitation of A. Cells from triplicate samples were scored for thepresence of VP2 in these samples.

FIG. 12 depicts the inhibitory properties of Retro-2^(cycl) analogs. Allcompounds were tested against JCPyV at 25 uM in 0.04% DMSO. The dashedline indicates the level of inhibition by Retro-2^(cycl).

DEFINITIONS Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

As used herein, “alkyl” refers to a radical of a straight-chain orbranched saturated hydrocarbon group having from 1 to 10 carbon atoms(“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 9 carbonatoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1to 7 carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl grouphas 1 to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkylgroup has 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, analkyl group has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments,an alkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In someembodiments, an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). Insome embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. Unless otherwise specified, each instance of an alkylgroup is independently unsubstituted (an “unsubstituted alkyl”) orsubstituted (a “substituted alkyl”) with one or more substituents. Incertain embodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl(e.g., —CH₃). In certain embodiments, the alkyl group is a substitutedC₁₋₁₀ alkyl.

“Perhaloalkyl” is a substituted alkyl group as defined herein whereinall of the hydrogen atoms are independently replaced by a halogen, e.g.,fluoro, bromo, chloro, or iodo. In some embodiments, the alkyl moietyhas 1 to 8 carbon atoms (“C₁₋₈ perhaloalkyl”). In some embodiments, thealkyl moiety has 1 to 6 carbon atoms (“C₁₋₆ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 4 carbon atoms (“C₁₋₄perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 3 carbonatoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In some embodiments, all of thehydrogen atoms are replaced with fluoro. In some embodiments, all of thehydrogen atoms are replaced with chloro. Examples of perhaloalkyl groupsinclude —CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 10 carbon atoms and one ormore double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C₂₋₁₀ alkenyl”).In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms(“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has 2 to 7carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenylgroup has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, analkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In someembodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”).In some embodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”).The one or more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 10 carbon atoms and one ormore triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀ alkynyl”).In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C₂₋₉alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms(“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has 2 to 7carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynyl group has2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, an alkynylgroup has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In some embodiments, analkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”). In someembodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃ alkynyl”).In some embodiments, an alkynyl group has 2 carbon atoms (“C₂ alkynyl”).The one or more carbon-carbon triple bonds can be internal (such as in2-butynyl) or terminal (such as in 1-butynyl). Examples of C₂₋₄ alkynylgroups include, without limitation, ethynyl (C₂), 1-propynyl (C₃),2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examplesof C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkynyl groups aswell as pentynyl (C₅), hexynyl (C₆), and the like. Additional examplesof alkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is a substituted C₂₋₁₀ alkynyl.

As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbonatoms (“C₃₋₁₀ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 7 ring carbon atoms (“C₃₋₇ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃),cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl(C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and thelike. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₀carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₀ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is an unsubstituted C₃₋₁₀ cycloalkyl.In certain embodiments, the cycloalkyl group is a substituted C₃₋₁₀cycloalkyl.

As used herein, “heterocyclyl” or “heterocyclic” refers to a radical ofa 3- to 14-membered nonaromatic ring system having ring carbon atoms and1 to 4 ring heteroatoms, wherein each heteroatom is independentlyselected from nitrogen, oxygen, and sulfur (“3-14 memberedheterocyclyl”). In heterocyclyl groups that contain one or more nitrogenatoms, the point of attachment can be a carbon or nitrogen atom, asvalency permits. A heterocyclyl group can either be monocyclic(“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) ortricyclic system (“tricyclic heterocyclyl”)), and can be saturated orcan contain one or more carbon carbon double or triple bonds.Heterocyclyl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heterocyclyl” also includes ring systems whereinthe heterocyclyl ring, as defined above, is fused with one or morecarbocyclyl groups wherein the point of attachment is either on thecarbocyclyl or heterocyclyl ring, or ring systems wherein theheterocyclyl ring, as defined above, is fused with one or more aryl orheteroaryl groups, wherein the point of attachment is on theheterocyclyl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heterocyclylring system. Unless otherwise specified, each instance of heterocyclylis independently unsubstituted (an “unsubstituted heterocyclyl”) orsubstituted (a “substituted heterocyclyl”) with one or moresubstituents. In certain embodiments, the heterocyclyl group is anunsubstituted 3-14 membered heterocyclyl. In certain embodiments, theheterocyclyl group is a substituted 3-14 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 membered nonaromaticring system having ring carbon atoms and 1-4 ring heteroatoms, whereineach heteroatom is independently selected from nitrogen, oxygen, andsulfur (“5-10 membered heterocyclyl”). In some embodiments, aheterocyclyl group is a 5-8 membered nonaromatic ring system having ringcarbon atoms and 1-4 ring heteroatoms, wherein each heteroatom isindependently selected from nitrogen, oxygen, and sulfur (“5-8 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6membered nonaromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing 1 heteroatom include, withoutlimitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-memberedheterocyclyl groups containing 1 heteroatom include, without limitation,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl,dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.Exemplary 5-membered heterocyclyl groups containing 2 heteroatomsinclude, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.Exemplary 5-membered heterocyclyl groups containing 3 heteroatomsinclude, without limitation, triazolinyl, oxadiazolinyl, andthiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1heteroatom include, without limitation, piperidinyl, tetrahydropyranyl,dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, piperazinyl,morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclylgroups containing 2 heteroatoms include, without limitation,triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1heteroatom include, without limitation, azepanyl, oxepanyl andthiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbonatoms and zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

“Aralkyl” is a subset of “alkyl” and refers to an alkyl group, asdefined herein, substituted by an aryl group, as defined herein, whereinthe point of attachment is on the alkyl moiety.

As used herein, “heteroaryl” refers to a radical of a 5-14 memberedmonocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromaticring system (e.g., having 6, 10, or 14 π electrons shared in a cyclicarray) having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen and sulfur (“5-14 membered heteroaryl”). Inheteroaryl groups that contain one or more nitrogen atoms, the point ofattachment can be a carbon or nitrogen atom, as valency permits.Heteroaryl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heteroaryl” includes ring systems wherein theheteroaryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the point of attachment is on theheteroaryl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heteroaryl ringsystem. “Heteroaryl” also includes ring systems wherein the heteroarylring, as defined above, is fused with one or more aryl groups whereinthe point of attachment is either on the aryl or heteroaryl ring, and insuch instances, the number of ring members designates the number of ringmembers in the fused polycyclic (aryl/heteroaryl) ring system.Polycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-membered heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

“Heteroaralkyl” is a subset of “alkyl” and refers to an alkyl group, asdefined herein, substituted by a heteroaryl group, as defined herein,wherein the point of attachment is on the alkyl moiety.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl moieties) as herein defined.

As used herein, the term “saturated” refers to a ring moiety that doesnot contain a double or triple bond, i.e., the ring contains all singlebonds.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, and alkynylene is the divalent moiety ofalkynyl.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). For purposes of this invention,heteroatoms such as nitrogen, oxygen, and sulfur may have hydrogensubstituents and/or any suitable substituent as described herein whichsatisfy the valencies of the heteroatoms and results in the formation ofa stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—PSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₄ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, ortwo R^(aa) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR)N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee),—SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —S(═O)R^(ee),—Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂, —C(═O)SR^(ee),—C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee), —P(═O)(R^(ee))₂,—OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or twogeminal R^(dd) substituents can be joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl, C₁perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl,3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R^(if) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

As used herein, the term “hydroxyl” or “hydroxy” refers to the group—OH. The term “substituted hydroxyl” or “substituted hydroxyl,” byextension, refers to a hydroxyl group wherein the oxygen atom directlyattached to the parent molecule is substituted with a group other thanhydrogen, and includes groups selected from —OR^(aa), —ON(R^(bb))₂,—OC(═O)SR^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂,—OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂,—OS(═O)R^(aa), —OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃,—OP(═O)₂R^(aa), —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —OP(═O)₂N(R^(bb))₂,and —OP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein.

As used herein, the term “thiol” or “thio” refers to the group —SH. Theterm “substituted thiol” or “substituted thio,” by extension, refers toa thiol group wherein the sulfur atom directly attached to the parentmolecule is substituted with a group other than hydrogen, and includesgroups selected from —SR^(aa), —S═SR^(cc), —SC(═S)SR^(aa),—SC(═O)SR^(aa), —SC(═O)OR^(aa), and —SC(═O)R^(aa), wherein R^(aa) andR^(cc) are as defined herein.

As used herein, the term, “amino” refers to the group —NH₂. The term“substituted amino,” by extension, refers to a monosubstituted amino, adisubstituted amino, or a trisubstituted amino, as defined herein.

As used herein, the term “monosubstituted amino” refers to an aminogroup wherein the nitrogen atom directly attached to the parent moleculeis substituted with one hydrogen and one group other than hydrogen, andincludes groups selected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))^(N)(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), —R^(bb) andR^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

As used herein, the term “disubstituted amino” refers to an amino groupwherein the nitrogen atom directly attached to the parent molecule issubstituted with two groups other than hydrogen, and includes groupsselected from —N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

As used herein, the term “trisubstituted amino” refers to an amino groupwherein the nitrogen atom directly attached to the parent molecule issubstituted with three groups, and includes groups selected from—N(R^(bb))₃ and —N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as definedherein.

As used herein, the term “acyl” refers a group wherein the carbondirectly attached to the parent molecule is sp² hybridized, and issubstituted with an oxygen, nitrogen or sulfur atom, e.g., a groupselected from ketones (—C(═O)R^(aa)), carboxylic acids (—CO₂H),aldehydes (—CHO), esters (—CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)),amides (—C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), andimines (—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)),C(═NR^(bb))N(R^(bb))₂), wherein R^(aa) and R^(bb) are as defined herein.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro,F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

As used herein, a “counterion” is a negatively charged group associatedwith a positively charged quarternary amine in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa),—C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂,—SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂,—C(═O)SR^(cc), —C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(cc)groups attached to an N atom are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, andwherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined above.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamate, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBDT-moc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is asulfur protecting group (also referred to as a “thiol protectinggroup”). Sulfur protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The invention is notintended to be limited in any manner by the above exemplary listing ofsubstituents.

Other Definitions

As used herein, the term “salt” or “pharmaceutically acceptable salt”refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, Berge et al., describes pharmaceutically acceptable salts indetail in J. Pharmaceutical Sciences (1977) 66:1-19.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, andaryl sulfonate.

As used herein, the term “prodrug” means a compound that can hydrolyze,oxidize, or otherwise react under biological conditions (e.g., in vitroor in vivo enzymatic conditions) to provide a pharmacologically activecompound. In certain cases, a prodrug has improved physical and/ordelivery properties over the parent compound. Prodrugs are typicallydesigned to enhance pharmacologically, pharmaceutically and/orpharmacokinetically based properties associated with the parentcompound. The advantage of a prodrug can lie in its physical properties,such as enhanced water solubility for parenteral administration atphysiological pH compared to the parent compound, or it enhancesabsorption from the digestive tract, or it may enhance drug stabilityfor long-term storage.

“Tautomer” includes two or more interconvertable compounds resultingfrom at least one formal migration of a hydrogen atom and at least onechange in valency (e.g., a single bond to a double bond, a triple bondto a single bond, or vice versa). The exact ratio of the tautomersdepends on several factors, including temperature, solvent, and pH.Tautomerizations (i.e., the reaction providing a tautomeric pair) may becatalyzed by acid or base. Exemplary tautomerizations includeketo-to-enol; amide-to-imide; lactam-to-lactim; enamine-to-imine; andenamine-to- (a different) enamine tautomerizations.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example mammals (e.g., primates (e.g., cynomolgusmonkeys, rhesus monkeys); commercially relevant mammals such as cattle,pigs, horses, sheep, goats, cats, and/or dogs), and/or birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys). In certain embodiments, the non-human animal is a mammal. Thenon-human animal may be a male or female and at any stage ofdevelopment. A non-human animal may be a transgenic animal.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from the specified condition (e.g., a pathogenicinfection) which reduces the severity of the condition or retards orslows the progression of the condition (“therapeutic treatment”), andalso contemplates an action that occurs before a subject begins tosuffer from the specified condition and which inhibits or reduces theseverity of the condition (“prophylactic treatment”).

In general, the “effective amount” of a compound refers to an amountsufficient to elicit the desired biological response, i.e., treating thecondition (e.g., infection). As will be appreciated by those of ordinaryskill in this art, the effective amount of a compound of the inventionmay vary depending on such factors as the desired biological endpoint,the pharmacokinetics of the compound, the disease being treated, themode of administration, and the age, health, and condition of thesubject. For example, the effective amount of a compound withanti-infective activity is the amount that results in a sufficientconcentration to kill the pathogen (e.g., virus), or to reduce theinfectivity of the pathogen (e.g., virus). An effective amountencompasses therapeutic and prophylactic treatment.

As used herein “infectivity” refers to the degree of pathogenicity of apathogen (virus) as indicated by case fatality rates and/or the abilityof the pathogen (e.g., virus) to invade the tissues of the subject;e.g., the ability of the pathogen (e.g., virus) to cause an infection.To reduce the infectivity refers to a reduction of this pathogeniccapacity of the pathogen (e.g., virus).

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment of the condition (e.g., infection)or to delay or minimize one or more symptoms associated with thecondition. A therapeutically effective amount of a compound means anamount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of condition, or enhances the therapeutic efficacy of anothertherapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent thecondition (e.g., infection, e.g., to prevent its recurrence), or one ormore symptoms associated with the condition. A prophylacticallyeffective amount of a compound means an amount of a therapeutic agent,alone or in combination with other agents, which provides a prophylacticbenefit in the prevention of the condition. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

As used herein, use of the phrase “at least one instance” refers to 1,2, 3, or 4 instances, but also encompasses a range, e.g., for example,from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from3 to 4 instances, inclusive.

As used herein, a “pure” compound indicates that the isolated compoundis substantially free of other compounds (contaminants). “Substantiallyfree” in this context indicates the compound comprises less than 15%,less than 10%, less than 9%, less than 8%, less than 7%, less than 6%,less than 5%, less than 4%, less than 3%, less than 2%, less than 1%,less than 0.5%, less than 0.1%, e.g., less than between about 0.1% toabout 10%, of other compounds and/or contaminants as determinedanalytically, e.g., by NMR spectroscopy. In certain embodiments,compounds of Formula (II) are pure and isolated, alone or present in apharmaceutical composition, and are substantially free of compounds ofFormula (I).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

As generally described herein, the present invention is based on thediscovery that the imine2-(((5-methylthiophen-2-yl)methylene)amino)-N-phenylbenzamide (RETRO-2)and the corresponding cyclized product,2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one(Retro-2^(cycl)) are inhibitors of polyomavirus (JCV, BKV, and SV40)infectivity. RETRO-2 rapidly converts to Retro-2^(cycl) in solution, andit is likely that the active compound against polyomavirus infectivityis Retro-2^(cycl). It is envisioned that varying substitution at the5-position of the thiophenyl ring and/or substitution on one or both ofthe phenyl rings may lead to other potent inhibitors of polyomaviralinfection. It is further envisioned that such compounds may also exhibitgood activity against other conditions, e.g., other pathogenicconditions associated with endosomal trafficking or sorting.

In one aspect, provided are compounds of Formula (I) and (II):

and pharmaceutically acceptable salts, tautomers, prodrugs, andstereoisomers thereof;wherein:

X is O, S, or NH;

each instance of R¹ is independently halo, —NO₂, —CN, —SCN, —OR^(A1),—SR^(A1), —N(R^(A1))₂, —C(═O)R^(A1), —OC(═O)R^(A1), —SC(═O)R^(A1),—NR^(A1)C(═O)R^(A1), —S(═O)₂R^(A1), substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl; each instance of R^(A1) isindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted carbocyclyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted hydroxyl,substituted or unsubstituted amino, substituted or unsubstituted thiol,an oxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to a sulfur atom, or a nitrogenprotecting group when attached to a nitrogen atom, or two R^(A1) groupsare joined to form a substituted or unsubstituted heterocyclic orheteroaryl ring;

or two R¹ groups are joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring;

R^(N) is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl;

-   -   each instance of R³ is independently halo, —NO₂, —CN, —SCN,        —OR^(A3), —SR^(A3), —N(R^(A3))², —C(═O)R^(A3), —OC(═O)R^(A3),        —SC(═O)R^(A3), —NR^(A3)C(═O)R^(A3), —S(═O)₂R^(A3), substituted        or unsubstituted alkyl, substituted or unsubstituted alkenyl,        substituted or unsubstituted alkynyl, substituted or        unsubstituted carbocyclyl, substituted or unsubstituted        heterocyclyl, substituted or unsubstituted aryl, and substituted        or unsubstituted heteroaryl,

each instance of R^(A3) is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, substitutedor unsubstituted thiol, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom, or twoR^(A3) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring;

or two R³ groups are joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring;

R⁴ is hydrogen, substituted or unsubstituted alkyl, or an nitrogenprotecting group;

corresponds to a single or double bond;

p is 0, 1, or 2; and

m is 0, 1, 2, 3, or 4.

Compounds of Formula (I) and (II) are envisioned useful in the treatmentand prevention of a viral infection and/or a pathogenic conditionassociated with aberrant endosomal trafficking, as further describedherein.

Various Embodiments of Compounds of Formula (I) and (II)

As generally defined above for compounds of Formula (I) and (II), X isO, S, or NH. In certain embodiments, X is O. In certain embodiments, Xis S. In certain embodiments, X is NH.

As generally defined above for compounds of Formula (I) and (II), eachinstance of R¹ is independently halo, —NO₂, —CN, —SCN, —OR^(A1),—SR^(A1), —N(R^(A1))₂, —C(═O)R^(A1), —OC(═O)R^(A1), —SC(═O)R^(A1),—NR^(A1)C(═O)R^(A1), —S(═O)₂R^(A1), substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, wherein each instance of R^(A1)is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, substituted orunsubstituted thiol, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom, or twoR^(A1) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring, or two R¹ groups are joined to form asubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted carbocyclic, or substituted orunsubstituted heterocyclic ring.

In certain embodiments, at least one instance of R¹ is halo, e.g.,fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I).

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted alkyl, e.g., substituted or unsubstituted C₁₋₆alkyl,substituted or unsubstituted C₁₋₂alkyl, substituted or unsubstitutedC₂₋₃alkyl, substituted or unsubstituted C₃₋₄alkyl, substituted orunsubstituted C₄₋₅alkyl, substituted or unsubstituted C₅₋₆alkyl,substituted or unsubstituted C₁alkyl, substituted or unsubstitutedC₂alkyl, substituted or unsubstituted C₃alkyl, substituted orunsubstituted C₄alkyl, substituted or unsubstituted C₅alkyl, orsubstituted or unsubstituted C₆alkyl. In certain embodiments, R¹ ismethyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄),tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), or n-hexyl (C₆).

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted alkenyl, e.g., substituted or unsubstituted C₂₋₆alkenyl,substituted or unsubstituted C₂₋₃alkenyl, substituted or unsubstitutedC₃₋₄alkenyl, substituted or unsubstituted C₄₋₅alkenyl, substituted orunsubstituted C₅₋₆alkenyl, substituted or unsubstituted C₂alkenyl,substituted or unsubstituted C₃alkenyl, substituted or unsubstitutedC₄alkenyl, substituted or unsubstituted C₅alkenyl, or substituted orunsubstituted C₆alkenyl. In certain embodiments, R¹ is substituted orunsubstituted allyl (C₃).

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted alkynyl, e.g., substituted or unsubstituted C₂₋₆alkynyl,substituted or unsubstituted C₂₋₃alkynyl, substituted or unsubstitutedC₃₋₄alkynyl, substituted or unsubstituted C₄₋₅alkynyl, substituted orunsubstituted C₅₋₆alkynyl, substituted or unsubstituted C₂alkynyl,substituted or unsubstituted C₃alkynyl, substituted or unsubstitutedC₄alkynyl, substituted or unsubstituted C₅alkynyl, or substituted orunsubstituted C₆alkynyl. In certain embodiments, R¹ is substituted orunsubstituted acetylene (C₂) or substituted or unsubstituted propargyl(C₃).

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted carbocyclyl, e.g., substituted or unsubstitutedC₃₋₆carbocyclyl, substituted or unsubstituted C₃₋₄carbocyclyl,substituted or unsubstituted C₄₋₅carbocyclyl, or substituted orunsubstituted C₅₋₆carbocyclyl.

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted heterocyclyl, e.g., substituted or unsubstituted 3- to6-membered heterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl.

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted aryl, e.g., substituted or unsubstituted phenyl.

In certain embodiments, at least one instance of R¹ is substituted orunsubstituted heteroaryl, e.g., substituted or unsubstituted 5- to6-membered heteroaryl.

In certain embodiments, at least one instance of R¹ is —NO₂. In certainembodiments, R¹ is —CN. In certain embodiments, R¹ is —SCN.

In certain embodiments, at least one instance of R¹ is —OR^(A1), e.g.,wherein R^(A1) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or an oxygen protecting group.

In certain embodiments, at least one instance of R¹ is —SR^(A1), e.g.,wherein R^(A1) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or a sulfur protecting group.

In certain embodiments, at least one instance of R¹ is —N(R^(A1))₂,e.g., wherein each instance of R^(A1) is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group, or two R^(A1) groups are joinedto form a substituted or unsubstituted heterocyclic or heteroaryl ring.

In certain embodiments, at least one instance of R¹ is —C(═O)R^(A1),e.g., wherein R^(A1) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R¹ is —OC(═O)R^(A1),e.g., wherein R^(A1) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R¹ is —SC(═O)R^(A1),e.g., wherein R^(A1) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R¹ is—NR^(A1)C(═O)R^(A1), e.g., wherein R^(A2) is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, orsubstituted or unsubstituted thiol, or R^(A1) is a nitrogen protectinggroup when attached to the nitrogen atom.

In certain embodiments, at least one instance of R¹ is —S(═O)₂R^(A1),e.g., wherein R^(A1) is substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted carbocyclyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted hydroxyl,substituted or unsubstituted amino, or substituted or unsubstitutedthiol.

In any of the above described embodiments, in certain instances, R^(A1)is hydrogen.

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted alkyl, e.g., substituted orunsubstituted C₁₋₆alkyl, substituted or unsubstituted C₁₋₂alkyl,substituted or unsubstituted C₂₋₃ alkyl, substituted or unsubstitutedC₃₋₄alkyl, substituted or unsubstituted C₄₋₅alkyl, substituted orunsubstituted C₅₋₆alkyl, substituted or unsubstituted C₁alkyl,substituted or unsubstituted C₂alkyl, substituted or unsubstitutedC₃alkyl, substituted or unsubstituted C₄alkyl, substituted orunsubstituted C₅alkyl, or substituted or unsubstituted C₆alkyl. Incertain embodiments, R^(A1) is methyl (C₁), ethyl (C₂), n-propyl (C₃),isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl(C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl (C₅),3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆).

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted alkenyl, e.g., substituted orunsubstituted C₂₋₆alkenyl, substituted or unsubstituted C₂₋₃alkenyl,substituted or unsubstituted C₃₋₄alkenyl, substituted or unsubstitutedC₄₋₅alkenyl, substituted or unsubstituted C₅₋₆alkenyl, substituted orunsubstituted C₂alkenyl, substituted or unsubstituted C₃alkenyl,substituted or unsubstituted C₄alkenyl, substituted or unsubstitutedC₅alkenyl, or substituted or unsubstituted C₆alkenyl. In certainembodiments, R^(A1) is substituted or unsubstituted allyl (C₃).

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted alkynyl, e.g., substituted orunsubstituted C₂₋₆alkynyl, substituted or unsubstituted C₂₋₃alkynyl,substituted or unsubstituted C₃₋₄alkynyl, substituted or unsubstitutedC₄₋₅alkynyl, substituted or unsubstituted C₅₋₆alkynyl, substituted orunsubstituted C₂alkynyl, substituted or unsubstituted C₃alkynyl,substituted or unsubstituted C₄alkynyl, substituted or unsubstitutedC₅alkynyl, or substituted or unsubstituted C₆alkynyl. In certainembodiments, R^(A1) is substituted or unsubstituted acetylene (C₂) orsubstituted or unsubstituted propargyl (C₃).

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted carbocyclyl, e.g., substituted orunsubstituted C₃₋₆carbocyclyl, substituted or unsubstitutedC₃₋₄carbocyclyl, substituted or unsubstituted C₄₋₅ carbocyclyl, orsubstituted or unsubstituted C₅₋₆ carbocyclyl.

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted heterocyclyl, e.g., substituted orunsubstituted 3- to 6-membered heterocyclyl, substituted orunsubstituted 3- to 4-membered heterocyclyl, substituted orunsubstituted 4- to 5-membered heterocyclyl, or substituted orunsubstituted 5- to 6-membered heterocyclyl.

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted aryl, e.g., substituted or unsubstitutedphenyl.

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted heteroaryl, e.g., substituted orunsubstituted 5- to 6-membered heteroaryl.

In any of the above described embodiments, in certain instances, R^(A1)is substituted or unsubstituted hydroxyl, substituted or unsubstitutedamino, substituted or unsubstituted thiol, an oxygen protecting groupwhen attached to an oxygen atom, a sulfur protecting group when attachedto a sulfur atom, or a nitrogen protecting group when attached to anitrogen atom.

In any of the above described embodiments, in certain instances, twoR^(A1) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring, e.g., joined to form a substituted orunsubstituted heterocyclic or heteroaryl ring, e.g., substituted orunsubstituted 3- to 6-membered heterocyclyl, substituted orunsubstituted 3- to 4-membered heterocyclyl, substituted orunsubstituted 4- to 5-membered heterocyclyl, or substituted orunsubstituted 5- to 6-membered heterocyclyl, or an substituted orunsubstituted 5- to 6-membered heteroaryl.

In certain embodiments, two R¹ groups are joined to form a substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted carbocyclic, or substituted orunsubstituted heterocyclic ring. Thus, for example, in any of theembodiments described herein, wherein p is 2, two R¹ groups vicinal toeach other may be joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring. Variousbicyclic Ring C systems are contemplated from the joining of two vicinalR¹ groups.

In certain embodiments, two R¹ groups are joined to form a substitutedor unsubstituted carbocyclyl, e.g., substituted or unsubstitutedC₃₋₆carbocyclyl, substituted or unsubstituted C₃₋₄carbocyclyl,substituted or unsubstituted C₄₋₅ carbocyclyl, or substituted orunsubstituted C₅₋₆ carbocyclyl.

In certain embodiments, two R¹ groups are joined to form a substitutedor unsubstituted heterocyclyl, e.g., substituted or unsubstituted 3- to6-membered heterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl.

In certain embodiments, two R¹ groups are joined to form a substitutedor unsubstituted aryl, e.g., substituted or unsubstituted phenyl.

In certain embodiments, two R¹ groups are joined to form a substitutedor unsubstituted heteroaryl, e.g., substituted or unsubstituted 5- to6-membered heteroaryl.

As generally defined above for compounds of Formula (I) and (II), p is0, 1, or 2, and refers to the number of substituents (or lack ofsubstituents when p is 0) attached to Ring C:

In certain embodiments, p is 0, and Ring C is an unsubstituted ring ofthe formula:

In certain embodiments, p is 1, and Ring C is a monosubstituted ring,e.g., of the formula:

In certain embodiments, p is 2, and Ring C is a disubstituted ring,e.g., of the formula:

As generally defined above for compounds of Formula (I) and (II), R^(N)is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl.

In certain embodiments, R^(N) is hydrogen.

In certain embodiments, R^(N) is substituted or unsubstituted alkyl,e.g., substituted or unsubstituted C₁₋₆alkyl, substituted orunsubstituted C₁₋₂alkyl, substituted or unsubstituted C₂₋₃alkyl,substituted or unsubstituted C₃₋₄alkyl, substituted or unsubstitutedC₄₋₅alkyl, substituted or unsubstituted C₅₋₆alkyl, substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(N) is methyl (C₁), ethyl (C₂),n-propyl (C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl(C₄), iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅),neopentyl (C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl(C₆).

In certain embodiments, R^(N) is substituted or unsubstituted alkenyl,e.g., substituted or unsubstituted C₂₋₆alkenyl, substituted orunsubstituted C₂₋₃alkenyl, substituted or unsubstituted C₃₋₄alkenyl,substituted or unsubstituted C₄₋₅alkenyl, substituted or unsubstitutedC₅₋₆ alkenyl, substituted or unsubstituted C₂alkenyl, substituted orunsubstituted C₃alkenyl, substituted or unsubstituted C₄alkenyl,substituted or unsubstituted C₅alkenyl, or substituted or unsubstitutedC₆alkenyl. In certain embodiments, R^(N) is substituted or unsubstitutedallyl (C₃).

In certain embodiments, R^(N) is substituted or unsubstituted alkynyl,e.g., substituted or unsubstituted C₂₋₆alkynyl, substituted orunsubstituted C₂₋₃alkynyl, substituted or unsubstituted C₃₋₄alkynyl,substituted or unsubstituted C₄₋₅alkynyl, substituted or unsubstitutedC₅₋₆alkynyl, substituted or unsubstituted C₂alkynyl, substituted orunsubstituted C₃alkynyl, substituted or unsubstituted C₄alkynyl,substituted or unsubstituted C₅alkynyl, or substituted or unsubstitutedC₆alkynyl. In certain embodiments, R^(N) is substituted or unsubstitutedacetylene (C₂) or substituted or unsubstituted propargyl (C₃).

In certain embodiments, R^(N) is substituted or unsubstitutedcarbocyclyl, e.g., substituted or unsubstituted C₃₋₆carbocyclyl,substituted or unsubstituted C₃₋₄carbocyclyl, substituted orunsubstituted C₄₋₅ carbocyclyl, or substituted or unsubstituted C₅₋₆carbocyclyl.

In certain embodiments, R^(N) is substituted or unsubstitutedheterocyclyl, e.g., substituted or unsubstituted 3- to 6-memberedheterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl.

In certain embodiments, R^(N) is substituted or unsubstituted aryl,e.g., substituted or unsubstituted phenyl.

In certain embodiments, R^(N) is substituted or unsubstitutedheteroaryl, e.g., substituted or unsubstituted 5- to 6-memberedheteroaryl.

In certain specific embodiments, wherein R^(N) is a substituted alkyl,substituted alkenyl, substituted alkynyl, or substituted aryl group,R^(N) comprises Ring A directly attached to the parent moiety (wherein Lis a bond) or attached via a linker group (wherein L¹ is substituted orunsubstituted alkylene, substituted or unsubstituted alkenylene, orsubstituted or unsubstituted alkynylene):

each instance of R² is independently halo, —NO₂, —CN, —SCN, —OR^(A2),—SR^(A2), —N(R^(A2))₂, —C(═O)R^(A2), —OC(═O)R^(A2), —SC(═O)R^(A2),—NR^(A2)C(═O)R^(A2), —S(═O)₂R^(A2), substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl,

each instance of R^(A2) is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, substitutedor unsubstituted thiol, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom, or twoR^(A2) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring;

or two R² groups are joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring; and

-   -   n is 0, 1, 2, 3, 4 or 5.

It is understood that for such compounds comprising Ring A, n refers tothe number of substituents (or lack of substituents when n is 0)attached to Ring A:

In certain embodiments, n is 0, and Ring A is an unsubstituted ring ofthe formula:

In certain embodiments, n is 1, and Ring A is a monosubstituted ring,e.g., of the formula:

In certain embodiments, n is 2, and Ring A is a disubstituted ring,e.g., of the formula:

In certain embodiments, n is 3, and Ring A is a trisubstituted ring,e.g., of the formula:

In certain embodiments, n is 4, and Ring A is a tetrasubstituted ring,e.g., of the formula:

In certain embodiments, n is 5, and Ring A is a pentasubstituted ring ofthe formula:

In certain embodiments, at least one instance of R² is halo, e.g.,fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I).

In certain embodiments, L¹ is a bond.

In certain embodiments, L¹ is substituted or unsubstituted alkylene,e.g., substituted or unsubstituted C₁₋₆alkylene, substituted orunsubstituted C₁₋₂alkylene, substituted or unsubstituted C₁₋₃alkylene,substituted or unsubstituted C₂₋₃alkylene, substituted or unsubstitutedC₃₋₄alkylene, substituted or unsubstituted C₄₋₅alkylene, substituted orunsubstituted C₅₋₆alkylene, substituted or unsubstituted C₁alkylene,substituted or unsubstituted C₂alkylene, substituted or unsubstitutedC₃alkylene, substituted or unsubstituted C₄alkylene, substituted orunsubstituted C₅alkylene, or substituted or unsubstituted C₆alkylene. Incertain embodiments, L¹ is methylene (C₁), ethylene (C₂), n-propylene(C₃), n-butylene (C₄), n-pentylene (C₅), or n-hexylene (C₆). In certainembodiments, L¹ is —CH(CH₃)— having (R) or (S) stereochemistry.

In certain embodiments, L¹ is substituted or unsubstituted alkenylene,e.g., substituted or unsubstituted C₂₋₆alkenylene, substituted orunsubstituted C₂₋₃alkenylene, substituted or unsubstitutedC₃₋₄alkenylene, substituted or unsubstituted C₄₋₅alkenylene, substitutedor unsubstituted C₅₋₆alkenylene, substituted or unsubstitutedC₂alkenylene, substituted or unsubstituted C₃alkenylene, substituted orunsubstituted C₄alkenylene, substituted or unsubstituted C₅alkenylene,or substituted or unsubstituted C₆alkenylene.

In certain embodiments, L is substituted or unsubstituted alkynylene,e.g., substituted or unsubstituted C₂₋₆alkynylene, substituted orunsubstituted C₂₋₃alkynylene, substituted or unsubstitutedC₃₋₄alkynylene, substituted or unsubstituted C₄₋₅alkynylene, substitutedor unsubstituted C₅₋₆alkynylene, substituted or unsubstitutedC₂alkynylene, substituted or unsubstituted C₃alkynylene, substituted orunsubstituted C₄alkynylene, substituted or unsubstituted C₅alkynylene,or substituted or unsubstituted C₆alkynylene.

In certain embodiments, at least one instance of R² is substituted orunsubstituted alkyl, e.g., substituted or unsubstituted C₁₋₆alkyl,substituted or unsubstituted C₁₋₂alkyl, substituted or unsubstitutedC₂₋₃ alkyl, substituted or unsubstituted C₃₋₄alkyl, substituted orunsubstituted C₄₋₅alkyl, substituted or unsubstituted C₅₋₆alkyl,substituted or unsubstituted C₁alkyl, substituted or unsubstitutedC₂alkyl, substituted or unsubstituted C₃alkyl, substituted orunsubstituted C₄alkyl, substituted or unsubstituted C₅alkyl, orsubstituted or unsubstituted C₆alkyl. In certain embodiments, R² ismethyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄),tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), or n-hexyl (C₆).

In certain embodiments, at least one instance of R² is substituted orunsubstituted alkenyl, e.g., substituted or unsubstituted C₂₋₆alkenyl,substituted or unsubstituted C₂₋₃alkenyl, substituted or unsubstitutedC₃₋₄alkenyl, substituted or unsubstituted C₄₋₅alkenyl, substituted orunsubstituted C₅₋₆alkenyl, substituted or unsubstituted C₂alkenyl,substituted or unsubstituted C₃alkenyl, substituted or unsubstitutedC₄alkenyl, substituted or unsubstituted C₅alkenyl, or substituted orunsubstituted C₆alkenyl. In certain embodiments, R² is substituted orunsubstituted allyl (C₃).

In certain embodiments, at least one instance of R² is substituted orunsubstituted alkynyl, e.g., substituted or unsubstituted C₂₋₆alkynyl,substituted or unsubstituted C₂₋₃alkynyl, substituted or unsubstitutedC₃₋₄alkynyl, substituted or unsubstituted C₄₋₅alkynyl, substituted orunsubstituted C₅₋₆alkynyl, substituted or unsubstituted C₂alkynyl,substituted or unsubstituted C₃alkynyl, substituted or unsubstitutedC₄alkynyl, substituted or unsubstituted C₅alkynyl, or substituted orunsubstituted C₆alkynyl. In certain embodiments, R² is substituted orunsubstituted acetylene (C₂) or substituted or unsubstituted propargyl(C₃).

In certain embodiments, at least one instance of R² is substituted orunsubstituted carbocyclyl, e.g., substituted or unsubstitutedC₃₋₆carbocyclyl, substituted or unsubstituted C₃₋₄carbocyclyl,substituted or unsubstituted C₄₋₅ carbocyclyl, or substituted orunsubstituted C₅₋₆ carbocyclyl.

In certain embodiments, at least one instance of R² is substituted orunsubstituted heterocyclyl, e.g., substituted or unsubstituted 3- to6-membered heterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl.

In certain embodiments, at least one instance of R² is substituted orunsubstituted aryl, e.g., substituted or unsubstituted phenyl.

In certain embodiments, at least one instance of R² is substituted orunsubstituted heteroaryl, e.g., substituted or unsubstituted 5- to6-membered heteroaryl.

In certain embodiments, at least one instance of R² is —NO₂. In certainembodiments, at least one instance of R² is —CN. In certain embodiments,at least one instance of R² is —SCN.

In certain embodiments, at least one instance of R² is —OR^(A2), e.g.,wherein R^(A2) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or an oxygen protecting group.In certain embodiments, R2 is —OR^(A2) wherein R^(A2) is substituted orunsubstituted alkyl.

In certain embodiments, at least one instance of R² is —SR^(A2), e.g.,wherein R^(A2) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or a sulfur protecting group.

In certain embodiments, at least one instance of R² is —N(R^(A2))₂,e.g., wherein each instance of R^(A2) is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group, or two R^(A2) groups are joinedto form a substituted or unsubstituted heterocyclic or heteroaryl ring.

In certain embodiments, at least one instance of R² is —C(═O)R^(A2),e.g., wherein R^(A2) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R² is —OC(═O)R^(A2),e.g., wherein R^(A2) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R² is —SC(═O)R^(A2),e.g., wherein R^(A2) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R² is—NR^(A2)C(═O)R^(A2), e.g., wherein R^(A2) is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, orsubstituted or unsubstituted thiol, or R^(A2) is a nitrogen protectinggroup when attached to the nitrogen atom.

In certain embodiments, at least one instance of R² is —S(═O)₂R^(A2),e.g., wherein R^(A2) is substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted carbocyclyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted hydroxyl,substituted or unsubstituted amino, or substituted or unsubstitutedthiol.

In any of the above described embodiments, in certain instances, R^(A2)is hydrogen.

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted alkyl, e.g., substituted orunsubstituted C₁₋₆alkyl, substituted or unsubstituted C₁₋₂alkyl,substituted or unsubstituted C₂₋₃ alkyl, substituted or unsubstitutedC₃₋₄alkyl, substituted or unsubstituted C₄₋₅alkyl, substituted orunsubstituted C₅₋₆alkyl, substituted or unsubstituted C₁alkyl,substituted or unsubstituted C₂alkyl, substituted or unsubstitutedC₃alkyl, substituted or unsubstituted C₄alkyl, substituted orunsubstituted C₅alkyl, or substituted or unsubstituted C₆alkyl. Incertain embodiments, R^(A2) is methyl (C₁), ethyl (C₂), n-propyl (C₃),isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl(C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl (C₅),3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆).

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted alkenyl, e.g., substituted orunsubstituted C₂₋₆alkenyl, substituted or unsubstituted C₂₋₃alkenyl,substituted or unsubstituted C₃₋₄alkenyl, substituted or unsubstitutedC₄₋₅alkenyl, substituted or unsubstituted C₅₋₆alkenyl, substituted orunsubstituted C₂alkenyl, substituted or unsubstituted C₃alkenyl,substituted or unsubstituted C₄alkenyl, substituted or unsubstitutedC₅alkenyl, or substituted or unsubstituted C₆alkenyl. In certainembodiments, R^(A2) is substituted or unsubstituted allyl (C₃).

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted alkynyl, e.g., substituted orunsubstituted C₂₋₆alkynyl, substituted or unsubstituted C₂₋₃alkynyl,substituted or unsubstituted C₃₋₄alkynyl, substituted or unsubstitutedC₄₋₅alkynyl, substituted or unsubstituted C₅₋₆alkynyl, substituted orunsubstituted C₂alkynyl, substituted or unsubstituted C₃alkynyl,substituted or unsubstituted C₄alkynyl, substituted or unsubstitutedC₅alkynyl, or substituted or unsubstituted C₆alkynyl. In certainembodiments, R^(A2) is substituted or unsubstituted acetylene (C₂) orsubstituted or unsubstituted propargyl (C₃).

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted carbocyclyl, e.g., substituted orunsubstituted C₃₋₆carbocyclyl, substituted or unsubstitutedC₃₋₄carbocyclyl, substituted or unsubstituted C₄₋₅ carbocyclyl, orsubstituted or unsubstituted C₅₋₆ carbocyclyl.

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted heterocyclyl, e.g., substituted orunsubstituted 3- to 6-membered heterocyclyl, substituted orunsubstituted 3- to 4-membered heterocyclyl, substituted orunsubstituted 4- to 5-membered heterocyclyl, or substituted orunsubstituted 5- to 6-membered heterocyclyl.

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted aryl, e.g., substituted or unsubstitutedphenyl.

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted heteroaryl, e.g., substituted orunsubstituted 5- to 6-membered heteroaryl.

In any of the above described embodiments, in certain instances, R^(A2)is substituted or unsubstituted hydroxyl, substituted or unsubstitutedamino, substituted or unsubstituted thiol, an oxygen protecting groupwhen attached to an oxygen atom, a sulfur protecting group when attachedto a sulfur atom, or a nitrogen protecting group when attached to anitrogen atom.

In any of the above described embodiments, in certain instances, twoR^(A2) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring, e.g., substituted or unsubstituted 3-to 6-membered heterocyclyl, substituted or unsubstituted 3- to4-membered heterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl, or an substituted or unsubstituted 5- to 6-memberedheteroaryl.

In certain embodiments, two R² groups are joined to form a substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted carbocyclic, or substituted orunsubstituted heterocyclic ring. Thus, for example, in any of theembodiments described herein, wherein n is 2, two R² groups vicinal toeach other may be joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring. Variousbicyclic Ring A systems are contemplated from the joining of two vicinalR² groups.

In certain embodiments, two R² groups are joined to form a substitutedor unsubstituted carbocyclyl, e.g., substituted or unsubstitutedC₃₋₆carbocyclyl, substituted or unsubstituted C₃₋₄carbocyclyl,substituted or unsubstituted C₄₋₅ carbocyclyl, or substituted orunsubstituted C₅₋₆ carbocyclyl.

In certain embodiments, two R² groups are joined to form a substitutedor unsubstituted heterocyclyl, e.g., substituted or unsubstituted 3- to6-membered heterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl.

In certain embodiments, two R² groups are joined to form a substitutedor unsubstituted aryl, e.g., substituted or unsubstituted phenyl.

In certain embodiments, two R² groups are joined to form a substitutedor unsubstituted heteroaryl, e.g., substituted or unsubstituted 5- to6-membered heteroaryl.

As generally defined above for compounds of Formula (I) and (II), m is0, 1, 2, 3, or 4, and refers to the number of substituents (or lack ofsubstituents when m is 0) attached to Ring B:

In certain embodiments, m is 0, and Ring B is unsubstituted, e.g., toprovide a Ring B of the formula:

In certain embodiments, m is 1, and Ring B is monosubstituted, e.g., toprovide a Ring B of the formula:

In certain embodiments, m is 2, and Ring B is disubstituted, e.g., toprovide a Ring B of the formula:

In certain embodiments, m is 3, and Ring B is trisubstituted, e.g., toprovide a Ring B of the formula:

In certain embodiments, m is 4, and Ring B is tetrasubstituted, e.g., toprovide a Ring B of the formula:

As generally defined above for compounds of Formula (I) and (II), eachinstance of R³ is independently halo, —NO₂, —CN, —SCN, —OR^(A3), —SR,—N(R^(A3))₂, —C(═O)R^(A3), —OC(═O)R^(A3), —SC(═O)R^(A3),—NR^(A3)C(═O)R^(A3), —S(═O)₂R^(A3), substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, wherein each instance of R^(A3)is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, substituted orunsubstituted thiol, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom, or twoR^(A3) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring; or two R³ groups are joined to form asubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted carbocyclic, or substituted orunsubstituted heterocyclic ring; and wherein m is 0, 1, 2, 3, or 4.

In certain embodiments, at least one instance of R³ is halo, e.g.,fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I).

In certain embodiments, at least one instance of R³ is substituted orunsubstituted alkyl, e.g., substituted or unsubstituted C₁₋₆alkyl,substituted or unsubstituted C₁₋₂alkyl, substituted or unsubstitutedC₂₋₃ alkyl, substituted or unsubstituted C₃₋₄alkyl, substituted orunsubstituted C₄₋₅alkyl, substituted or unsubstituted C₅₋₆alkyl,substituted or unsubstituted C₁alkyl, substituted or unsubstitutedC₂alkyl, substituted or unsubstituted C₃alkyl, substituted orunsubstituted C₄alkyl, substituted or unsubstituted C₅alkyl, orsubstituted or unsubstituted C₆alkyl. In certain embodiments, R³ ismethyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄),tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), or n-hexyl (C₆).

In certain embodiments, at least one instance of R³ is substituted orunsubstituted alkenyl, e.g., substituted or unsubstituted C₂₋₆alkenyl,substituted or unsubstituted C₂₋₃alkenyl, substituted or unsubstitutedC₃₋₄alkenyl, substituted or unsubstituted C₄₋₅alkenyl, substituted orunsubstituted C₅₋₆alkenyl, substituted or unsubstituted C₂alkenyl,substituted or unsubstituted C₃alkenyl, substituted or unsubstitutedC₄alkenyl, substituted or unsubstituted C₅alkenyl, or substituted orunsubstituted C₆alkenyl. In certain embodiments, R³ is substituted orunsubstituted allyl (C₃).

In certain embodiments, at least one instance of R³ is substituted orunsubstituted alkynyl, e.g., substituted or unsubstituted C₂₋₆alkynyl,substituted or unsubstituted C₂₋₃alkynyl, substituted or unsubstitutedC₃₋₄alkynyl, substituted or unsubstituted C₄₋₅alkynyl, substituted orunsubstituted C₅₋₆alkynyl, substituted or unsubstituted C₂alkynyl,substituted or unsubstituted C₃alkynyl, substituted or unsubstitutedC₄alkynyl, substituted or unsubstituted C₅alkynyl, or substituted orunsubstituted C₆alkynyl. In certain embodiments, R³ is substituted orunsubstituted acetylene (C₂) or substituted or unsubstituted propargyl(C₃).

In certain embodiments, at least one instance of R³ is substituted orunsubstituted carbocyclyl, e.g., substituted or unsubstitutedC₃₋₆carbocyclyl, substituted or unsubstituted C₃₋₄carbocyclyl,substituted or unsubstituted C₄₋₅ carbocyclyl, or substituted orunsubstituted C₅₋₆ carbocyclyl.

In certain embodiments, at least one instance of R³ is substituted orunsubstituted heterocyclyl, e.g., substituted or unsubstituted 3- to6-membered heterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl.

In certain embodiments, at least one instance of R³ is substituted orunsubstituted aryl, e.g., substituted or unsubstituted phenyl.

In certain embodiments, at least one instance of R³ is substituted orunsubstituted heteroaryl, e.g., substituted or unsubstituted 5- to6-membered heteroaryl.

In certain embodiments, at least one instance of R³ is —NO₂. In certainembodiments, at least one instance of R³ is —CN. In certain embodiments,at least one instance of R³ is —SCN. In certain embodiments, at leastone instance of R³ is —OR^(A3), e.g., wherein R^(A3) is hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or an oxygen protecting group.

In certain embodiments, at least one instance of R³ is —SR^(A3), e.g.,wherein R^(A3) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or a sulfur protecting group.

In certain embodiments, at least one instance of R³ is —N(R^(A3))₂,e.g., wherein each instance of R^(A3) is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group, or two R^(A3) groups are joinedto form a substituted or unsubstituted heterocyclic or heteroaryl ring.

In certain embodiments, at least one instance of R³ is —C(═O)R^(A3),e.g., wherein R^(A3) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R³ is —OC(═O)R^(A3),e.g., wherein R^(A3) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R³ is —SC(═O)R^(A3),e.g., wherein R^(A3) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedhydroxyl, substituted or unsubstituted amino, or substituted orunsubstituted thiol.

In certain embodiments, at least one instance of R³ is—NR^(A3)C(═O)R^(A3), e.g., wherein R^(A3) is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, orsubstituted or unsubstituted thiol, or R^(A3) is a nitrogen protectinggroup when attached to the nitrogen atom.

In certain embodiments, at least one instance of R³ is —S(═O)₂R^(A3),e.g., wherein R^(A3) is substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted carbocyclyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted hydroxyl,substituted or unsubstituted amino, or substituted or unsubstitutedthiol.

In any of the above described embodiments, in certain instances, R^(A3)is hydrogen.

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted alkyl, e.g., substituted orunsubstituted C₁₋₆alkyl, substituted or unsubstituted C₁₋₂alkyl,substituted or unsubstituted C₂₋₃ alkyl, substituted or unsubstitutedC₃₋₄alkyl, substituted or unsubstituted C₄₋₅alkyl, substituted orunsubstituted C₅₋₆alkyl, substituted or unsubstituted C₁alkyl,substituted or unsubstituted C₂alkyl, substituted or unsubstitutedC₃alkyl, substituted or unsubstituted C₄alkyl, substituted orunsubstituted C₅alkyl, or substituted or unsubstituted C₆alkyl. Incertain embodiments, R^(A3) is methyl (C₁), ethyl (C₂), n-propyl (C₃),isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl(C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl (C₅),3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆).

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted alkenyl, e.g., substituted orunsubstituted C₂₋₆alkenyl, substituted or unsubstituted C₂₋₃alkenyl,substituted or unsubstituted C₃₋₄alkenyl, substituted or unsubstitutedC₄₋₅alkenyl, substituted or unsubstituted C₅₋₆alkenyl, substituted orunsubstituted C₂alkenyl, substituted or unsubstituted C₃alkenyl,substituted or unsubstituted C₄alkenyl, substituted or unsubstitutedC₅alkenyl, or substituted or unsubstituted C₆alkenyl. In certainembodiments, R^(A3) is substituted or unsubstituted allyl (C₃).

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted alkynyl, e.g., substituted orunsubstituted C₂₋₆alkynyl, substituted or unsubstituted C₂₋₃alkynyl,substituted or unsubstituted C₃₋₄alkynyl, substituted or unsubstitutedC₄₋₅alkynyl, substituted or unsubstituted C₅₋₆alkynyl, substituted orunsubstituted C₂alkynyl, substituted or unsubstituted C₃alkynyl,substituted or unsubstituted C₄alkynyl, substituted or unsubstitutedC₅alkynyl, or substituted or unsubstituted C₆alkynyl. In certainembodiments, R^(A3) is substituted or unsubstituted acetylene (C₂) orsubstituted or unsubstituted propargyl (C₃).

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted carbocyclyl, e.g., substituted orunsubstituted C₃₋₆carbocyclyl, substituted or unsubstitutedC₃₋₄carbocyclyl, substituted or unsubstituted C₄₋₅ carbocyclyl, orsubstituted or unsubstituted C₅₋₆ carbocyclyl.

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted heterocyclyl, e.g., substituted orunsubstituted 3- to 6-membered heterocyclyl, substituted orunsubstituted 3- to 4-membered heterocyclyl, substituted orunsubstituted 4- to 5-membered heterocyclyl, or substituted orunsubstituted 5- to 6-membered heterocyclyl.

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted aryl, e.g., substituted or unsubstitutedphenyl.

In any of the above described embodiments, in certain instances, R^(A3)is substituted or unsubstituted heteroaryl, e.g., substituted orunsubstituted 5- to 6-membered heteroaryl. In any of the above describedembodiments, in certain instances, R^(A3) is substituted orunsubstituted hydroxyl, substituted or unsubstituted amino, substitutedor unsubstituted thiol, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom.

In any of the above described embodiments, in certain instances, twoR^(A3) groups are joined to form a substituted or unsubstitutedheterocyclic or heteroaryl ring, e.g., substituted or unsubstituted 3-to 6-membered heterocyclyl, substituted or unsubstituted 3- to4-membered heterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl, or an substituted or unsubstituted 5- to 6-memberedheteroaryl.

In certain embodiments, two R³ groups are joined to form a substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted carbocyclic, or substituted orunsubstituted heterocyclic ring. Thus, for example, in any of theembodiments described herein, wherein m is 2, two R³ groups vicinal toeach other may be joined to form a substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclic, or substituted or unsubstituted heterocyclic ring. Variousbicyclic Ring B systems are contemplated from the joining of two vicinalR³ groups.

In certain embodiments, two R³ groups are joined to form a substitutedor unsubstituted carbocyclyl, e.g., substituted or unsubstitutedC₃₋₆carbocyclyl, substituted or unsubstituted C₃₋₄carbocyclyl,substituted or unsubstituted C₄₋₅ carbocyclyl, or substituted orunsubstituted C₅₋₆ carbocyclyl.

In certain embodiments, two R³ groups are joined to form a substitutedor unsubstituted heterocyclyl, e.g., substituted or unsubstituted 3- to6-membered heterocyclyl, substituted or unsubstituted 3- to 4-memberedheterocyclyl, substituted or unsubstituted 4- to 5-memberedheterocyclyl, or substituted or unsubstituted 5- to 6-memberedheterocyclyl. In certain embodiments, two R³ groups are joined to form asubstituted or unsubstituted aryl, e.g., substituted or unsubstitutedphenyl.

In certain embodiments, two R³ groups are joined to form a substitutedor unsubstituted heteroaryl, e.g., substituted or unsubstituted 5- to6-membered heteroaryl. As generally defined above for compounds ofFormula (II), R⁴ is hydrogen, substituted or unsubstituted alkyl, or annitrogen protecting group, and

corresponds to a single or double bond.

In certain embodiments, R⁴ is hydrogen.

In certain embodiments, R⁴ is substituted or unsubstituted alkyl, e.g.,substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstitutedC₁₋₂alkyl, substituted or unsubstituted C₂₋₃alkyl, substituted orunsubstituted C₃₋₄alkyl, substituted or unsubstituted C₄₋₅alkyl,substituted or unsubstituted C₅₋₆alkyl, substituted or unsubstitutedC₁alkyl, substituted or unsubstituted C₂alkyl, substituted orunsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl, substitutedor unsubstituted C₅alkyl, or substituted or unsubstituted C₆alkyl.

In certain embodiments, R⁴ is an nitrogen protecting group.

In certain embodiments,

corresponds to a single bond. In certain embodiments,

corresponds to a double bond.

Combinations of the above described embodiments are furthercontemplated.

For example, in certain embodiments of Formula (I) and (II), wherein pis 1, provided is a compound of Formula (I-a), (I-b), (I-c), (II-a),(II-b), or (II-c):

or a pharmaceutically acceptable salt, tautomer, prodrug, orstereoisomer thereof. In certain embodiments, X is S. In certainembodiments,

corresponds to a double bond. In certain embodiments, R¹ is halo orsubstituted or unsubstituted C₁₋₆ alkyl. In certain embodiments, R¹ is—Br. In certain embodiments, R¹ is methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆). Incertain embodiments, R^(N) is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl. In certain embodiments, R^(N) is substituted orunsubstituted carbocyclyl. In certain embodiments, R^(N) is a group offormula

In certain embodiments, n is 0, 1, or 2. In certain embodiments, eachinstance of R² is independently halo, —NO₂, —OR^(A2). In certainembodiments, L¹ is a bond. In certain embodiments, L¹ is substituted orunsubstituted C₁alkylene, substituted or unsubstituted C₂alkylene,substituted or unsubstituted C₃alkylene. In certain embodiments, L¹ ismethylene (C₁), ethylene (C₂), n-propylene (C₃). In certain embodiments,L¹ is —CH(CH₃)— having (R) or (S) stereochemistry. In certainembodiments, m is 0 or 1. In certain embodiments, each instance of R³ isindependently halo or substituted or unsubstituted alkyl. In certainembodiments, R⁴ is hydrogen.

In certain embodiments of Formula (I) and (II), wherein R^(N) comprisesa Ring A, provided is a compound of Formula (I-d) or (II-d):

or a pharmaceutically acceptable salt, tautomer, prodrug, orstereoisomer thereof. In certain embodiments, X is S. In certainembodiments,

corresponds to a double bond. In certain embodiments, R¹ is halo orsubstituted or unsubstituted C₁₋₆ alkyl. In certain embodiments, R¹ is—Br. In certain embodiments, R¹ is methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆). Incertain embodiments, n is 0, 1, or 2. In certain embodiments, eachinstance of R² is independently halo, —NO₂, —OR^(A2). In certainembodiments, L¹ is a bond. In certain embodiments, L is substituted orunsubstituted C₁alkylene, substituted or unsubstituted C₂alkylene,substituted or unsubstituted C₃alkylene. In certain embodiments, L¹ ismethylene (C₁), ethylene (C₂), n-propylene (C₃). In certain embodiments,L¹ is —CH(CH₃)— having (R) or (S) stereochemistry. In certainembodiments, m is 0 or 1. In certain embodiments, each instance of R³ isindependently halo or substituted or unsubstituted alkyl. In certainembodiments, R⁴ is hydrogen.

In certain embodiments of Formula (I) and (II), wherein p is 1 and R^(N)comprises a Ring A, provided is a compound of Formula (I-e) and (II-e):

In certain embodiments of Formula (I) and (II), wherein p is 1, n is 0,1, or 2, and R^(N) comprises a Ring A, provided is a compound of any oneof the following Formula:

or a pharmaceutically acceptable salt, tautomer, prodrug, orstereoisomer thereof. In certain embodiments, X is S. In certainembodiments,

corresponds to a double bond. In certain embodiments, R¹ is halo orsubstituted or unsubstituted C₁₋₆ alkyl. In certain embodiments, R¹ is—Br. In certain embodiments, R¹ is methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆). Incertain embodiments, each instance of R² is independently halo, —NO₂,—OR^(A2). In certain embodiments, L¹ is a bond. In certain embodiments,L¹ is substituted or unsubstituted C₁alkylene, substituted orunsubstituted C₂alkylene, substituted or unsubstituted C₃alkylene. Incertain embodiments, L¹ is methylene (C₁), ethylene (C₂), n-propylene(C₃). In certain embodiments, L¹ is —CH(CH₃)— having (R) or (S)stereochemistry. In certain embodiments, m is 0 or 1. In certainembodiments, each instance of R³ is independently halo or substituted orunsubstituted alkyl. In certain embodiments, R⁴ is hydrogen.

Specific compounds of Formula (I) and (II) are further contemplatedherein.

For example, compounds of Formula (I), and pharmaceutically acceptablesalts, tautomers, prodrugs, and stereoisomers thereof, include, but arenot limited to:

However, in certain embodiments, the compound of Formula (I) is not2-(((5-methylthiophen-2-yl)methylene)amino)-N-phenylbenzamide orN-phenyl-2-((thiophen-2-ylmethylene)amino)benzamide:

Compounds of Formula (II), and pharmaceutically acceptable salts,tautomers, prodrugs, and stereoisomers thereof, include, but are notlimited to:

However, in certain embodiments, the compound of Formula (II) is not2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one or3-phenyl-2-(thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one:

Pharmaceutical Compositions

In still yet another aspect, provided are pharmaceutical compositionscomprising a compound of Formula (I) and (II), or a mixture thereof, ora pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomerthereof, and a pharmaceutically acceptable excipient. Compounds ofFormula (I) and (II) are also referred to herein as the “activeingredient(s).” In certain embodiments, the pharmaceutical compositioncomprises an effective amount of the active ingredient(s). In certainfurther embodiments, the pharmaceutical composition is useful as amedicament for the treatment and prevention of a viral infection and/ora pathogenic condition associated with aberrant endosomal trafficking.

Pharmaceutically acceptable excipients include any and all solvents,diluents or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. General considerations in theformulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21^(st) Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing the active ingredient(s) intoassociation with a carrier and/or one or more other accessoryingredients, and then, if necessary and/or desirable, shaping and/orpackaging the product into a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the activeingredient(s). The amount of the active ingredient(s) is generally equalto the dosage of the active ingredient(s) which would be administered toa subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. Pharmaceuticallyacceptable excipients used in the manufacture of the pharmaceuticalcomposition include inert diluents, dispersing and/or granulatingagents, surface active agents and/or emulsifiers, disintegrating agents,binding agents, preservatives, buffering agents, lubricating agents,and/or oils. Excipients such as cocoa butter and suppository waxes,coloring agents, coating agents, sweetening, flavoring, and perfumingagents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, etc., and combinationsthereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g. acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g.bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]),long chain amino acid derivatives, high molecular weight alcohols (e.g.stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate,ethylene glycol distearate, glyceryl monostearate, and propylene glycolmonostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene,polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer),carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium,powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acidesters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20],polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate[Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate[Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitanmonooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylenemonostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,polyethoxylated castor oil, polyoxymethylene stearate, and Solutol),sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether[Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate,triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate,oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68,Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride,benzalkonium chloride, docusate sodium, etc. and/or combinationsthereof.

Exemplary binding agents include starch (e.g. cornstarch and starchpaste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghattigum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum),and larch arabogalactan), alginates, polyethylene oxide, polyethyleneglycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes,water, alcohol, etc., and/or combinations thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary oils include, but are not limited to, butylstearate, caprylic triglyceride, capric triglyceride, cyclomethicone,diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil,octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the activeingredient(s)s, the liquid dosage forms may comprise inert diluentscommonly used in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can include adjuvants such as wettingagents, emulsifying and suspending agents, sweetening, flavoring, andperfuming agents. In certain embodiments for parenteral administration,the conjugates of the invention are mixed with solubilizing agents suchas Cremophor, alcohols, oils, modified oils, glycols, polysorbates,cyclodextrins, polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active ingredient(s).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient(s) is mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active ingredient(s) can be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient(s) can be admixed with at least oneinert diluent such as sucrose, lactose or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 0.01% to about 10% (w/w) active ingredient(s), although theconcentration of the active ingredient(s) can be as high as thesolubility limit of the active ingredient(s) in the solvent.Formulations for topical administration may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient(s) and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient(s) dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient(s) in the form of droplets ofa solution and/or suspension. Such formulations can be prepared,packaged, and/or sold as aqueous and/or dilute alcoholic solutionsand/or suspensions, optionally sterile, comprising the activeingredient(s), and may conveniently be administered using anynebulization and/or atomization device. Such formulations may furthercomprise one or more additional ingredients including, but not limitedto, a flavoring agent such as saccharin sodium, a volatile oil, abuffering agent, a surface active agent, and/or a preservative such asmethylhydroxybenzoate. The droplets provided by this route ofadministration may have an average diameter in the range from about 0.1to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient(s) and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered by rapid inhalation through the nasal passage from acontainer of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient(s), and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition can beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and maycontain, for example, 0.1 to 20% (w/w) active ingredient(s), the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising the active ingredient(s). Such powdered,aerosolized, and/or aerosolized formulations, when dispersed, may havean average particle and/or droplet size in the range from about 0.1 toabout 200 nanometers, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition can be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient(s) inan aqueous or oily liquid carrier. Such drops may further comprisebuffering agents, salts, and/or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient(s) in microcrystalline form and/or in a liposomalpreparation. Ear drops and/or eye drops are contemplated as being withinthe scope of this invention.

Compounds provided herein are typically formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular subject or organism will dependupon a variety of factors including the disease, disorder, or conditionbeing treated and the severity of the disorder; the activity of thespecific active ingredient(s) employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thesubject; the time of administration, route of administration, and rateof excretion of the specific active ingredient(s) employed; the durationof the treatment; drugs used in combination or coincidental with thespecific active ingredient(s) employed; and like factors well known inthe medical arts.

The active ingredient(s) provided herein can be administered by anyroute, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal,enteral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; and/or as an oral spray, nasal spray,and/or aerosol. In general the most appropriate route of administrationwill depend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),the condition of the subject (e.g., whether the subject is able totolerate oral administration), etc.

The exact amount of the active ingredient(s) required to achieve aneffective amount will vary from subject to subject, depending, forexample, on species, age, and general condition of a subject, severityof the side effects or disorder, identity of the particular compound(s),mode of administration, and the like. The desired dosage can bedelivered three times a day, two times a day, once a day, every otherday, every third day, every week, every two weeks, every three weeks, orevery four weeks. In certain embodiments, the desired dosage can bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations).

In certain embodiments, an effective amount of the active ingredient(s)for administration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about1000 mg, or about 100 mg to about 1000 mg, of an inventive compound perunit dosage form. It will be appreciated that dose ranges as describedherein provide guidance for the administration of providedpharmaceutical compositions to an adult. The amount to be administeredto, for example, a child or an adolescent can be determined by a medicalpractitioner or person skilled in the art and can be lower or the sameas that administered to an adult.

It will be also appreciated that the active ingredient(s), as describedherein, can be administered in combination with one or more additionalactive agents. The active ingredient(s) can be administered concurrentlywith, prior to, or subsequent to, one or more additional active agents.In general, each active ingredient(s) and additional active agent willbe administered at a particular dose and/or on a particular timeschedule. In will further be appreciated that the additional activeagent utilized in this combination can be administered together in asingle composition or administered separately in different compositions.The particular combination to employ in a regimen will take into accountcompatibility of the active ingredient(s), with the additional activeagent and/or the desired therapeutic effect to be achieved. In general,it is expected that additional active agents utilized in combination beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually. Theadditional active agents may improve the active ingredient(s)bioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. It willalso be appreciated that therapy employed may achieve a desired effectfor the same condition, e.g., anti-infective activity, and/or it mayachieve different effects (e.g., control of adverse side-effects).

Exemplary one or more additional active agents includes, but is notlimited to, anti-viral agents. In certain embodiments, the anti-viralagent is a virus receptor antagonist. In certain embodiments, the virusreceptor antagonist is an inhibitor of serotonin receptor. In certainembodiments, the anti-viral agent is an antibody, e.g., designed torecognize the virus and prevent initial infection.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.General considerations in the formulation and/or manufacture ofpharmaceutical compositions can be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005.

Still further encompassed by the invention are pharmaceutical kits.Pharmaceutical kits provided may comprise a pharmaceutical composition,as described herein, and a container (e.g., a vial, ampoule, bottle,syringe, and/or dispenser package, or other suitable container), andinstructions for use. In some embodiments, the kits may optionallyfurther include a second container comprising a suitable aqueous carrierfor dilution or suspension of the composition for preparation ofadministration to a subject. Instructions may provide, for example,instructions for dosage and administration, specialized instructions forparticular containers and/or systems for administration, and/orinstructions for use in combination with additional therapies.

Methods of Treatment and Use

The present invention is based, in part, on the discovery that the imine2-(((5-methylthiophen-2-yl)methylene)amino)-N-phenylbenzamide (RETRO-2)and the corresponding cyclized product,2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one(Retro-2^(cycl)), encompassed by Formula (I) or (II), respectively, areinhibitors of polyomavirus (e.g., JCV, BKV, and SV40) infectivity. Thus,in one aspect, the present invention provides use of compounds ofFormula (I) or (II) for treating and preventing viral infections, suchas polyomaviral infections. In certain embodiments, the compounds ofFormula (I) or (II) are useful in reducing infectivity.

RETRO-2 was initially described in Stechmann et al., Cell (2010)141:231-242 as an AB5 toxin inhibitor; thus novel compounds of Formula(I) and (II) are further contemplated useful in treating or preventingconditions caused by AB5 toxin, such as treating or preventing aninfection with a pathogen (e.g., bacteria) that secretes an AB5 toxin.Such AB5 toxins include, but are not limited to, the toxin ricin, Shigatoxin and Shiga-like toxins, cholera toxin, heat-labile enterotoxin,pertussis toxin, and subtilase cytotoxin.

Polyomaviruses traffic to the ER, but, unlike AB5 toxin, thepolyomavirus does not traffic to the ER via the Golgi apparatus, butappears to bypass the Golgi en route to the ER. See, e.g., Ewers et al.,Cold Spring Harb Perspect Biol (2011) 3:a004721. Data suggests that theRetro-2^(cycl) blocks one or more steps in this genomic trafficking. SeeFIG. 6, infra. The present invention further contemplates use ofcompounds of Formula (I) and (II) as inhibitors of pathogenic conditionsassociated with endosomal trafficking, e.g., conditions wherein apathogen uses a host cell's endosomal trafficking pathway.

Thus, in one aspect, the present invention provides a method of treatingor preventing a viral infection, the method comprising administering toa subject suffering from or likely to suffer from a viral infection aneffective amount of a compound of Formula (I) or (II), or a mixturethereof, or pharmaceutically acceptable salt, tautomer, prodrug, orstereoisomer thereof.

In another aspect, the present invention provides a method of treatingor preventing a pathogenic condition associated with endosomaltrafficking, the method comprising administering to a subject sufferingfrom or likely to suffer from the condition an effective amount of acompound of Formula (I) or (II), or a mixture thereof, orpharmaceutically acceptable salt, tautomer, prodrug, or stereoisomerthereof. In certain embodiments, the pathogenic condition is a bacterialinfection, a fungal infection, a parasitic infection, or a viralinfection. In certain embodiments, the pathogenic condition is abacterial infection. In certain embodiments, the pathogenic condition isa viral infection.

In yet another aspect, the present invention provides a method oftreating or preventing an infection by a pathogen that secretes AB5toxin, the method comprising administering to a subject suffering fromor likely to suffer from the infection an effective amount of a compoundof Formula (I) or (II), or a mixture thereof, or pharmaceuticallyacceptable salt, tautomer, prodrug, or stereoisomer thereof. In certainembodiments, the pathogen secreting an AB5 toxin is a bacteria, e.g., E.coli. In certain embodiments, the AB5 toxin is the toxin ricin, Shigatoxin, Shiga-like toxins, cholera toxin, heat-labile enterotoxin,pertussis toxin, or subtilase cytotoxin.

In yet another aspect, the present invention provides a method oftreating or preventing an infection by a pathogen that secretes a toxinthat relies on retrograde endosomal trafficking, the method comprisingadministering to a subject suffering from or likely to suffer from theinfection an effective amount of a compound of Formula (I) or (II), or amixture thereof, or pharmaceutically acceptable salt, tautomer, prodrug,or stereoisomer thereof.

Therapeutic and prophylactic methods of the above are contemplated.

For example, in certain embodiments, the methods as described herein aretherapeutic treatment methods, comprising administering to a subjectsuffering from the infection or condition an effective amount of acompound of Formula (I) or (II), or a mixture thereof, orpharmaceutically acceptable salt, tautomer, prodrug, or stereoisomerthereof.

Alternatively, in certain embodiments, the methods as described hereinare preventative methods, comprising administering to a subject likelyto suffer from the infection or condition an effective amount of acompound of Formula (I) or (II), or a mixture thereof, orpharmaceutically acceptable salt, tautomer, prodrug, or stereoisomerthereof.

In certain embodiments, the viral infection or pathogenic conditionrequires retrograde endosomal transport of the pathogen's (viral) genometo the cell nucleus. In certain embodiments, the compound of Formula (I)or (II), or a mixture thereof, or pharmaceutically acceptable salt,tautomer, prodrug, or stereoisomer thereof, inhibits retrogradeendosomal transport of the pathogen's (viral) genome to the cellnucleus. In certain embodiments, the pathogen is a virus. In certainembodiments, the pathogen is a polyomavirus.

In certain embodiments, the viral infection is an infection caused byhuman papillomavirus (HPV). In certain embodiments, the HPV is HPV-16 orHPV-18.

In certain embodiments, the viral infection is an infection caused byhuman immunodeficiency virus (HIV). In certain embodiments, the HIV isHIV type 1 (HIV-1).

In certain embodiments, the viral infection is an infection caused byinfluenza virus. In certain embodiments, the influenza virus isinfluenzavirus A, influenzavirus B, influenzavirus C, or humanparainfluenza virus. In certain embodiments, influenzavirus A is H1N1,H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, or H10N7.

In certain embodiments, the viral infection is an infection caused bypolyomavirus. In certain embodiments, the polyomavirus isJC-polyomavirus, BK-polyomavirus KI-polyomavirus, WU-polyomavirus,Merkel cell polyomavirus, HPyV6, HPyV7, trichodysplasiaspinulosa-associated polyomavirus, HPyV9, MW polyomavirus, or the monkeypolyomavirus SV40. In certain embodiments, the viral infection is aninfection in a human subject caused by human polyomavirus.

In certain embodiments, the viral infection is JCV, and the subjectfurther suffers from progressive multifocal leukoencephalopathy (PML).

In certain embodiments, the viral infection is BKV, and the subjectfurther suffers from kidney necrosis and/or polyomavirus-inducedneuropathy (PVN).

In certain embodiments, the viral infection is KIV or WUV, and thesubject further suffers from a respiratory tract infection.

In certain embodiments, the viral infection is MCV, and the subjectfurther suffers from Merkel cell carcinomas of the skin.

In certain embodiments, the viral infection is TSV, and the subjectfurther suffers from proliferative skin lesions.

In certain embodiments, the subject is human. In certain embodiments,the human subject is immusuppresed. In certain embodiments, the subjectis immunosuppressed due to an infection (e.g., HIV infection). Incertain embodiments, the subject is immunosuppressed due to anautoimmune disease (e.g., rheumatoid arthritis, multiple sclerosis). Incertain embodiments, the subject is immunosuppressed due to cancer orcancer treatment (e.g., upon treatment with immunomodulatory drugs). Incertain embodiments, the subject is immunosuppressed due to old age(e.g., older than 65 years old). In certain embodiments, the subject isimmunosuppressed due to organ transplantation.

EXAMPLES

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

Retro-2^(cycl) as an Inhibitor of Polyomavirus Infectivity

We discovered a small molecule that inhibits infection by two humanpolyomaviruses, JCV and BKV, and the monkey polyomavirus SV40, RETRO-2and Retro-2^(cycl). RETRO-2 forms the closed ring structureRetro-2^(cycl) rapidly in solution, so it is likely that Retro-2^(cycl)is the active form in cellulo and in vivo. As the compound interactswith host cell factors rather than the polyomavirus themselves, it isalso likely that escape mutants will arise less frequently. RETRO-2 andRetro-2^(cycl) and analogs thereof represent the first potentialtreatment that broadly inhibits polyomaviruses.

The viability of SVG-A or Vero cells were first tested after treatmentwith Retro-2^(cycl). See FIG. 2. Cells were treated with Retro-2^(cycl)at the indicated concentrations for 72 h before viability was assessedusing the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Asa control, cells were treated with either 0.04% DMSO or with 10% SDS.Error bars denote standard deviation. The data demonstratesRetro-2^(cycl) is effective at doses that do not negatively affect cellviability.

It was further found that Retro-2^(cycl) inhibits infectivity of JCV,BKV, or SV40 at differing concentrations. See FIG. 1. Cells wereinfected with the indicated concentration of virus for 1 h. Media wasthen added to these cells containing the indicated concentration ofRetro-2^(cycl) and cells were incubated for 72 h. Cells were thendetached from each well, fixed, permeabalized, and stained withmonoclonal antibody against VP1 that has been covalently labeled withAlexa Fluor 488. Infected cells were scored by flow cytometry, and werenormalized against an infected control that was treated with the vehicleDMSO. The DMSO concentration was 0.04% in all samples. Error bars denotestandard deviation. These data demonstrates Retro-2^(cycl) inhibitspolyomavirus in a dose dependent manner. As a negative control, Verocells were incubated with an Adenovirus Type 5 pseudovirus thatexpresses GFP in transduced cells (Ad5-GFP). Since Adenovirus does notundergo retrograde trafficking the golgi complex or endoplasmicreticulum, treatment with Retro-2^(cycl) was expected to have no effecton cellular transduction. These data demonstrates that Ad5-GFPtransduction is unaffected by Retro-2^(cycl) treatment.

Retro-2^(cycl) further prevents spread of JCV, BKV, or SV40 inestablished tissue culture infections. See FIG. 3. Cells were infectedwith JCV, BKV, or SV40 at an multiplicity of infection of approximately0.01 and were allowed to infect cells for 72 h. After 72 h, 100 μM ofRetro-2^(cycl) was added to each sample. Tissue culture media wasreplaced each day with fresh media containing Retro-2^(cycl). Replicateswere assayed at 3, 6, 9, and 12 days post infection and the percentageof infected cells was determined by flow cytometry. Error bars denotestandard deviation. The data demonstrates Retro-2^(cycl) reduces thespread of polyomavirus over time in tissue culture.

Treatment of infected cells with Retro-2^(cycl) also reduces the amountof infectious virons released into the tissue culture media. See FIG. 4.At 6, 9, and 12 days post infection, the tissue culture supernatant (ofFIG. 3) was removed, and 0.1 mL of each sample was added to uninfectedcells. After 72 h, these cells were assayed by flow cytometry todetermine the percentage of cells that are infected in each sample.Error bars denote standard deviation. The data demonstratesRetro-2^(cycl) treatment results in significantly less virus beingproduced in culture.

Retro-2^(cycl) also reduces cytopathic effect in SVG-A cells. See FIG.5. JCV infected SVG-A cells (of FIG. 3) at that were maintained for 12days with Retro-2^(cycl) or DMSO were detached and seeded into newtissue culture wells and were maintained in media containingRetro-2^(cycl) or DMSO. At 15 days post infection these cells wereimaged by phase contrast microscopy.

It was further found that Retro-2^(cycl) inhibits early steps ininfectious entry of JCV, BKV, or SV40. See FIG. 6. In order to determinewhich steps in viral entry are affected by Retro-2^(cycl) treatment ofcells, cells were incubated with JCV, BKV, or SV40 at 4 degrees for 1 hto prevent endocytosis. Cells were washed and then media lacking orcontaining Retro-2^(cycl) (0 h time point) was added and the cells wereincubated at 37 degrees. At the indicated time points, media was removedand replaced with media containing Retro-2^(cycl). Cells were thenincubated at 37 degrees for a total of 72 h and assayed by flowcytometry for the percentage of infected cells as compared to a vehicletreated negative control. Error bars denote standard deviation. The datademonstrates Retro-2^(cycl) loses most of its inhibitory effect after 12hours (14 hours for JCV), suggesting Retro-2^(cycl) blocks steps inviral trafficking.

It was also found that Retro-2^(cycl) treatment of cells does notprevent binding of Alexa Fluor 633 labeled JCV (JCV-633). See FIG. 7.SVG-A cells were pretreated with the 100 μM of Retro-2^(cycl) at 4degrees for 1 h prior to incubation with JCV-633 for 1 h at 4 degrees.Vero cells were pretreated with 100 μM of Retro-2^(cycl) at 4 degreesfor 1 h prior to incubation with BKV-633, SV40-633, or CTxB-488 for 1 hat 4 degrees Cells were then assayed by flow cytometry to determine thelevel of polyomavirus or toxin binding as compared to cells treated witha vehicle control.

Other analogs, such as the ethyl analog of Retro-2^(cycl), wereexplored. See FIG. 8. Cells were inoculated with JCV at an MOI ofapproximately 1 for 1 h at 37 degrees. After 1 h, media was addedcontaining analog compounds at 100 μM (FIG. 8) in a final DMSOconcentration of 0.04%. After 72 h, cells were assayed by flow cytometryfor infected cells and data was normalized to infected cells treatedwith the DMSO vehicle. Error bars denote standard deviation. The datademonstrate that Retro-2^(cycl) inhibit infection of cells bypolyomaviruses.

We questioned whether Retro-2^(cycl) prevents polyomavirus ERtrafficking. See FIGS. 10 and 11. Cells were chilled to 4 degrees C. andinoculated with labeled JCV. After inoculation, the cells were fixed at6 hpi, the ER was immunostained with an antibody to protein disulfideisomerase (an ER resident enzyme) and imaged by confocal microscopy. Thedata demonstrates that Retro-2^(cycl) treatment appears to decreaseco-localization of JCV with the ER. Alternatively cells were transfectedwith early endosomal and ER markers [Early endosome (Rab5-RFP) in green;Endoplasmic reticulum (CFP-HO) in blue; JCV in red]. Transfected cellswere then inoculated with JCV-633 and incubated for 6 h prior toconfocal imaging. This experiment further demonstrates thatRetro-2^(cycl) treatment reduced ER colocalization and increasesco-localization of JCV-633 with Rab5 endosomes, and thus prevents JCVfrom reaching the endoplasmic reticulum in cells (FIG. 10C). FIG. 10Adepicts the quantification of the data shown in FIG. 10B, demonstratingthat Retro-2^(cycl) decreases ER trafficking of JCV.

A Retrograde Trafficking Inhibitor of Ricin and Shiga-Like ToxinsInhibits Infection of Cells by Human and Monkey Polyomaviruses

Human polyomaviruses are widespread pathogens that establish persistentlifelong infections in their hosts. See, e.g., Jiang et al., J Virol(2009) 83: 1350-1358, Kean et al., PLoS Pathog (2009) 5: e1000363.Infection of immunocompetent individuals by polyomaviruses usuallyresults in asymptomatic infection, with occurrence of disease only inimmunosuppressed individuals. See, e.g., Jiang et al., J Virol (2009)83: 1350-1358. Two human polyomaviruses, JC polyomavirus (JCPyV) and BKpolyomavirus (BKPyV), establish persistent infections early in life andchronically infect cells of the kidney, urinary tract, tonsillar stromalcells, and bone marrow derived cells. See, e.g., Monaco et al., J Virol(1996) 70: 7004-7012; Monaco et al., J Virol (1998) 72: 9918-9923;Ferenczy et al., Clin Microbiol Rev (2012) 25: 471-506; Shinohara etal., J Med Virol (1993) 41: 301-305. The seroprevelence of JCPyV andBKPyV are 50% and 80% respectively. See, e.g., Chesters et al., J InfectDis (1983) 147: 676-684; Egli et al., J Infect Dis (2009) 199: 837-846;Knowles et al., J Med Virol (2003) 71: 115-123; Knowles et al., Adv ExpMed Biol (2006) 577: 19-45. It is likely that JCPyV and BKPyVpersistently replicate at low levels, as virus is sporadically detectedin the urine of 30% of individuals tested. See, e.g., Kitamura et al., JInfect Dis (1990) 161: 1128-1133; Yogo et al., J Virol (1990) 64:3139-3143; Kahan et al., Am J Clin Pathol (1980) 74: 326-332. Underconditions of immunosuppression, such as acquired immunodeficiencysyndrome (AIDS) or immunomodulatory therapy, increased replication ofJCPyV results in dissemination of virus to the central nervous system.See, e.g., Ferenczy et al., Clin Microbiol Rev (2012) 25: 471-506. Aftercrossing the blood brain barrier, lytic infection of oligodendrocytes byJCPyV results in the fatal demyelinating disease progressive multifocalleukoencephalopathy (PML). See, e.g., Stoner et al., Proc Natl Acad SciUSA (1986) 83: 2271-2275; Zurhein et al., Science (1965) 148: 1477-1479;Khalili et al., Mult Scler (2006) 12: 133-142. The incidence of PML inAIDS patients is between 3% and 5% and the incidence in patientsreceiving immunomodulatory therapies is between 0.2% and 0.4%. See,e.g., Ferenczy et al., Clin Microbiol Rev (2012) 25: 471-506.BKPyV-associated disease is most often seen in the context of renaltransplantation, where immunosuppressive therapies result in increasedreplication of BKPyV in the transplanted kidney leading to hemorrhagiccystitis and polyomavirus nephropathy (PVN). See, e.g., Boothpur et al.,J Clin Virol (2010) 47: 306-312. The incidence of PVN in transplantrecipients can be as high as 10%, often resulting in loss of thetransplanted kidney. See, e.g., Binet et al., Transplantation (1999) 67:918-922.

There are no effective anti-viral therapies to combat infection by thesepolyomaviruses. Polyomaviruses are small, non-enveloped, double strandedDNA viruses that replicate in the nucleus. Despite being structurallysimple viruses, polyomaviruses utilize a complex and incompletelyunderstood entry process in order to effect nuclear trafficking. Afterbinding to cellular receptors on the cell surface, polyomaviruses enterthe classical endocytic pathway, first trafficking in early and thenlate endosomes. See, e.g., Engel et al., J Virol. (2011) 85:4198-4211;Liebl et al., J Virol (2006) 80: 4610-4622; Querbes et al., J Virol(2006) 80: 9402-9413; Qian et al., PLoS Pathog (2009) 5: e1000465. Fromearly or late endosomes, all polyomaviruses studied to date then undergoretrograde trafficking to the ER, where they interact with and utilizeER resident host chaperones to promote infection. See, e.g., Sapp etal., Virology (2009) 384: 400-409. Interaction of virions with ERchaperones results in partial disassembly of the capsid andretrotranslocation of the virion into the cytosol. See, e.g., Schelhaaset al., Cell (2007) 131: 516-529; Lilley et al., J Virol (2006) 80:8739-8744; Magnuson et al., Mol Cell (2005) 20: 289-300; Rainey-Bargeret al., J Virol (2007) 81: 12996-13004; Goodwin et al., mBio (2011) 2:3e00101-11.

Despite the importance on ER trafficking, the specific host cellularmachinery utilized to promote ER targeting of virions remains unclear.Many polyomaviruses, including BKPyV, simian virus 40 (SV40), and somestrains of JCPyV utilize gangliosides as receptors for binding and entryinto cells. See, e.g., Low et al., J Virol (2006) 80: 1361-1366;Campanero-Rhodes et al., Journal of Virology (2007) 81: 12846-12858;Tsai et al., EMBO (2003) J 22: 4346-4355; Gorelik et al., J Infect Dis(2011) 204: 103-114. Similarly, bacterial and plant toxins such asricin, shiga-like toxins (SLTs), and cholera toxin β subunit (CTxB),bind glycolipids and undergo retrograde trafficking, resulting inaccumulation of toxins in the Golgi apparatus and ER. See, e.g.,Johannes et al., Cell (2008) 135: 1175-1187. It is likely that bindingto glycolipids plays an important role in recruiting host cell machinerythat results in accumulation of polyomaviruses and toxins in the ER.See, e.g., Ewers et al., Cold Spring Harb Perspect Biol 2011; doi:10.1101/cshperspect.a004721; Sandvig et al., International Journal ofMedical Microbiology (2004) 293: 483-490. Studies have demonstrated thatbacterial toxins usurp a large number of host factors to facilitateretrograde trafficking, including vesicle coat proteins such as theretromer complex, clathrin and epsinR, and the vesicle fusion proteinssyntaxin 5 and 6. See, e.g., Johannes et al., Cell (2008) 135:1175-1187. Whether polyomaviruses also utilize similar host factors toundergo ER trafficking remains unclear.

Treatment of cells with brefeldin A (BFA), an inhibitor that blocks COPImediated Golgi to ER trafficking, inhibits retrograde trafficking oftoxins and protects cells from intoxication. See, e.g., Mallard et al.,The Journal of Cell Biology (1998) 143: 973-990; Donta et al., TheJournal of Infectious Diseases (1995) 171: 721-724. Polyomavirusinfectivity is also inhibited by BFA treatment and also results indecreased accumulation of virions in the ER. See, e.g., Richards et al.,Mol Biol Cell (2002) 13: 1750-1764; Norkin et al., J Virol (2002) 76:5156-5166; Gilbert et al., Journal of Virology (2004) 78: 12259-12267;Damm et al., J Cell Biol (2005) 168: 477-488; Nelson et al., Virology(2012) 428: 30-40. However, BFA treatment of cells results insignificant morphological and physiological changes, and it is unclearwhether BFA treatment inhibits retrograde trafficking of polyomavirusesand toxins by similar mechanisms. See, e.g., Lippincott-Schwartz et al.,Cell (1989) 56: 801-813; Huotari et al., The EMBO Journal (2011) 30:3481-3500; Misumi et al., The Journal of Biological Chemistry (1986)261: 11398-11403; Low et al., The Journal of Biological Chemistry (1991)266: 17729-17732. Additionally, no polyomavirus has been found toassociate with the Golgi apparatus, suggesting that there aredifferences in pathways or kinetics used by these molecules to targetthe ER.

In this study we demonstrate that a small molecule, referred to asRetro-2^(cycl) potently inhibits infection of tissue culture cells byJCPyV, BKPyV, and SV40. Retro-2^(cycl) was previously identified by highthroughput screening for small molecules that would inhibit intoxicationof host cells by ricin and SLTs. See, e.g., Stechmann et al., Cell(2010) 141: 231-242. This compound was shown to prevent retrogradetrafficking from endosomes to the Golgi apparatus and subsequent stepsin toxin trafficking, thus preventing intoxication of host cells. Ratherthan binding to the toxins, Retro-2^(cycl) appears to interfere withretrograde trafficking of cargo by interaction with an unidentifiedcellular host factor. Unlike BFA, Retro-2^(cycl) does not alter cellularcompartment morphology, as the only cellular factors to have alteredcellular distribution after Retro-2^(cycl) treatment were syntaxin 5 and6. Furthermore, Retro-2^(cycl) is non toxic when administered to mice atlevels that protect against ricin challenge, suggesting that thiscompound is well suited for drug development. In this study, wedemonstrate that Retro-2^(cycl) inhibits polyomavirus infection byinterfering with ER trafficking. This suggests that polyomaviruses andricin/SLT toxins share a dependency on similar host factors forsuccessful intracellular trafficking. Further optimization of thiscompound should result in the development of effective antiviralcompounds that inhibit polyomavirus replication and reducepolyomavirus-associated diseases.

Inhibition of Polyomavirus Infection in a Dose Dependent Manner

We purchased Retro-2 (later determined to be Retro-2^(cycl), seefollowing discussion) from Chembridge and pre-treated cells permissivefor each type of virus (SVG-A for JCPyV, and Vero for SV40 and BKPyV),with non-toxic concentrations of Retro-2^(cycl). Cells were theninoculated with JCpyV, BKpyV or SV40 in the presence of this drug andinfected cells were scored by flow cytometry. Treatment with thecompound purchased resulted in a dose dependent decrease in thepercentage of cells expressing the viral late capsid gene VP1 comparedto a vehicle control, with a calculated EC₅₀ of 28.4 μM, 61.2 μM, and58.6 μM for JCPyV, BKPyV, and SV40 respectively. See FIG. 1. Thecompound purchased also decreased expression of the viral early genelarge T antigen with similar EC₅₀ values (data not shown). As a control,we pre-treated Vero cells with the compound purchased and inoculatedthem with an adenovirus pseudovirus packaging a GFP reporter plasmid(Ad5-GFP), a virus that does not utilize retrograde trafficking forproductive infection. See, e.g., Meier et al., J Gene Med (2004) 6 Suppl1: S152-163. Treatment did not inhibit transduction, suggesting that theeffect of the compound on infection is specific to virions that undergoretrograde trafficking. See FIG. 1B.

Reduction of Viral Spread in Established Tissue Culture Infections

Since most individuals are already persistently infected with JCPyV orBKPyV prior to immunosuppression, we sought to determine whether thecompound purchased could prevent viral spread in tissue cultures duringestablished infections. We infected SVG-A or Vero cells with JCPyV,BKPyV or SV40 at a low multiplicity of infectivity (MOI) of 0.01.Following one round of productive infection, 100 μM of the compound wasadded to these cells, which were then maintained in media containing thecompound, re-feeding samples daily with fresh media containing 100 μM ofthe compound. Treatment of cells with the compound resulted in asignificant reduction in the percentage of infected cells as compared tosamples treated to vehicle control. This effect was most striking at 12days post infection, where the compound severely diminished viral spreadfor SV40 (84% inhibition), BKPyV (89%), and JCPyV (90.5%). See FIG. 3 Toexamine whether treatment of these cultures with the compound inhibitedvirion production, the supematants from each time point were used tore-infect naïve cells and the percentage of infected cells weredetermined. This assay showed that cultures that were previously treatedwith the compound produced significantly less infectious virions intothe tissue culture supernatant. See FIG. 4. These results demonstratethat the compound decreases cell-to-cell spread of polyomaviruses inpreviously infected cultures.

The Bioactive Compound is a Dihydroquinazolinone Derivative of Retro-2

Condensation of 2-aminobenzanilide with4-methyl-2-thiophencarboxaldehyde following the method reported inPCT/IB2009/006334 yielded a mixture of two products both having theexpected molecular weight for Retro-2. See FIG. 8A. The two compoundswere separated and independently characterized. One product was Retro-2as indicated by a characteristic singlet at 11.0 ppm for the imineproton (data not shown). The second product was revealed to be adihydroquinazolinone by x-ray diffraction crystallography, referred toas Retro-2^(cycl). See FIG. 8B. The spectroscopic data for the compoundpurchased from Chembridge was identical to that of thedihydroquinazolinone derivative, Retro-2^(cycl), and not of thestructure reported for the acylic compound, Retro-2. To test theinhibitory effects of these compounds on JCPyV infection, wepre-incubated SVG-A cells with 100 μM of Retro-2 or Retro-2^(cycl), andinfected cells with JCpyV and scored infections by flow cytometry. As apositive control, we also treated cells with brefeldin A (BFA), a drugthat inhibits COP1 mediated retrograde trafficking and has previouslybeen shown to prevent ER trafficking of polyomaviruses. See, e.g.,Richards et al., Mol Biol Cell (2002) 13: 1750-1764. Surprisingly, bothRetro-2 and Retro-2^(cycl) inhibited polyomavirus infection with similarefficacy. See FIG. 8C. Treatment of cells with 20 ng/mL of brefeldin Aresulted in a 96% reduction in infectivity. See FIG. 8C.

Imine species similar in structure to Retro-2 are commonly invoked asmechanistic intermediates in the formation of dihydroquinazolinones,which suggests that the two chemical species could interconvert in theinfectivity assay thus accounting for their similar biologicalactivities. See, e.g., Narasimhulu et al., Tetrahedron (2011) 67:9627-9634; Prakash et al., Org Lett (2012) 14: 1896-1899; Sharma et al.,J Org Chem (2012) 77: 929-937; Shaterian et al., SyntheticCommunications (2010) 40: 1231-1242; Wang et al., Chinese ChemicalLetters (2011) 22: 1423-1426. Accordingly, we found that treatment ofRetro-2 with scandium (III) triflate, in methanol resulted in rapidconversion to the Retro-2^(cycl). In aqueous media, Retro-2 most likelycyclizes into Retro-2^(cycl) as well. During the preparation of thismanuscript, another group reported that Retro-2 slowly cyclizes inmethanol to Retro-2^(cycl), and that Retro-2^(cycl) was able to protectcells from Ricin and Stx2.

Despite the observation that Retro-2 spontaneously cyclizes in polarprotic solvents, it was still unclear whether the biologically activecompound was Retro-2 or Retro-2^(cycl). Treatment of Retro-2^(cycl) withsodium cyanoborohydride in methanol slowly produced a reduced species(Retro-2^(red)), which indicated that cyclization is reversible and thatRetro-2 and Retro-2^(cycl) exist in equilibrium. See FIG. 8D. However,despite their structural similarities, Retro-2^(red) is significantlyless active than Retro-2. See FIG. 8C. We also prepared a Retro-2regioisomer wherein the carboxamide and imine moieties are metasubstituted (Retro-2^(meta)) therefore precluding cyclization. See FIG.8E. This compound was also significantly less active and served as auseful negative control in subsequent experiments. Together, the lack ofbiological activity intrinsic to Retro-2^(red) as well as Retro-2^(meta)has lead us to the conclusion that the chemical species responsible forinhibition of polyomavirus infection is in fact thedihydroquinazolinone, Retro-2^(cycl).

Retro-2^(cycl) Inhibits Polyomavirus at Early Time Points DuringInfection

We hypothesized that Retro-2^(cycl) inhibits polyomavirus trafficking tothe ER, since the drug inhibits retrograde trafficking of ricin and SLTsfrom endosomes to the Golgi apparatus. SVG-A or Vero cells weresynchronously infected with JCPyV, BKPyV, or SV40, and Retro-2^(cycl)was added to cells at the indicated time points and the amount ofinfectivity was compared to vehicle control. The results show thataddition of Retro-2^(cycl) at time points up to 4 hours post infection(hpi) significantly reduces infectivity, with a progressive loss in itsinhibitory effect at time points after 6 hpi. See FIG. 6. By 18 hpi,greater than 80% of infectivity has been regained. The kinetics of theseeffects are consistent with previous reports showing that polyomavirusescolocalize with ER markers at 6-16 hpi See, e.g., Nelson et al.,Virology (2012) 428: 30-40. Therefore, the protective effect ofRetro-2^(cycl) is lost following time points consistent withlocalization to the ER, suggesting that this compound acts to limitpolyomavirus trafficking to this compartment.

Retro-2^(cycl) does not Prevent Attachment or Entry of JCPyV into HostCells

To test whether Retro-2^(cycl) inhibits steps upstream of endosomal toER trafficking, we pretreated SVG-A or Vero cells with Retro-2^(cycl)and assayed for attachment using Alexa fluor 633 labeled JCPyV, BKPyV,or SV40. Pretreatment of cells for 1 h with 100 μM Retro-2^(cycl)resulted similar levels of binding of JCpyV or CTxB to cells compared tothe vehicle control treated cells. See FIG. 9. We did see slightdecreases in the binding of BKpyV and SV40 to cells, but significantvirions still bound to cells and the protective effect of Retro-2^(cycl)was not lost at these time points, suggesting that this was not themajor mechanism by which Retro-2^(cycl) protects cells from infection.We next sought to determine whether Retro-2^(cycl) could preventendocytosis of virions. To assay for endocytosis, we pre-treated cellswith Retro-2^(cycl), a DMSO vehicle control, chloropromazine (aninhibitor of JCpyV endocytosis), or nystatin (an inhibitor of BKpyV,SV40, and CTxB endocytosis) prior to inoculation with Alexa fluor 488labeled JCPyV, BKPyV, SV40, or CTxB. After washing, these samples werewarmed to allow for synchronous endocytosis and fixed at 2 h. Todetermine whether virions were internalized or still present on theplasma membrane, we imaged these samples (pseudocolored green), thenadded trypan blue and re-imaged the samples (pseudocolored red). Trypanblue is a membrane impermeable dye that quenches the fluorescence ofvirions on the plasma membrane. Therefore, virions that have enteredcells still fluoresce after addition of trypan blue and arepseudocolored yellow. This experiment demonstrates that Retro-2^(cycl)treatment does not inhibit endocytosis of JCpyV, BKpyV, SV40, or CTxB.See FIG. 9. In contrast, chloropromazine inhibits endocytosis of JCpyVand nystatin treatment of cells inhibits endocytosis of BKpyV, SV40, andCTxB. Thus Retro-2^(cycl) does not inhibit binding or endocytosis ofpolyomaviruses into host cells.

Retro-2^(cycl) Reduces Retrograde Trafficking of Polyomaviruses and CTxB

Since Retro-2^(cycl) prevents endosomal to Golgi trafficking of ricin,SLTs, and CTxB, we sought to determine whether treatment of cells withRetro-2^(cycl) would interfere with delivery of virions or CTxB to theER. Cells were pre-incubated with the Retro-2^(cycl) or a vehiclecontrol and then inoculated with purified JCPyV, BKPyV, SV40 or CTxB.Cells were then fixed at 8 hpi (3 hpi for cholera toxin) andimmunostained for colocalization with the ER protein, protein disulfideisomerase (PDI). Treatment of cells with the vehicle control or thenon-inhibitory Retro-2 analog, Retro-2^(meta), results in significantcolocalization between polyomaviruses or CTxB with the ER. See FIG. 10A.Conversely, treatment of cells with BFA or Retro-2^(cycl) significantlyreduces colocalization of JCpyV, BKpyV, or CTxB, demonstrating thatthese compounds inhibit ER accumulation of virions and toxins. SV40 andPDI colocalization was reduced, although this reduction was notsignificant. To visualize this colocalization, cells were inoculatedwith Alexa fluor 488 labeled JCpyV, BKpyV, or SV40 and the ER wasimmunostained with an antibody to PDI. In DMSO or Retro-2^(meta) treatedsamples, colocalization between virions and PDI is visible. See FIG.10B. At 3 hpi, perinuclear accumulation of CTxB is visible in DMSO orRetro-2^(meta) treated cells. Treatment of cells with BFA orRetro-2^(cycl) results in less colocalization of virions with PDI, anddisperses the perinuclear accumulation of CTxB. Taken together, theseresults demonstrate that Retro-2^(cycl) significantly reduces ERtrafficking of polyomaviruses.

Retro-2^(cycl) Prevents Exposure of the Viral Minor Capsid Proteins

Upon trafficking to the ER, polyomaviruses interact with host cellchaperones for productive infection. See, e.g., Tsai et al., Curr TopMicrobiol Immunol (2010) 343: 177-194. These interactions result inisomeraziation of inter-pentameric disulfide bonds that normallycross-link the capsid together resulting in partial uncoating of thevirion. As a result, the normally sequestered minor capsid protein VP2is externalized and is accessible to antibodies. See, e.g., Norkin etal., J Virol (2002) 76: 5156-5166. Since Retro-2^(cycl) reduces deliveryof virions to the ER, we examined whether Retro-2^(cycl) will alsoreduce exposure of the minor capsid proteins of these viruses. Weinoculated cells with JCPyV, BKPyV, or SV40 in the presence of 100 μMRetro-2^(cycl) or vehicle control. In cells treated with the DMSOcontrol at 10 hpi, discrete punctae corresponding to VP2 are visualizedin a perinuclear region of the cells. See FIG. 11. In contrast,treatment of cells with Brefeldin A results in a significant reductionin the number of cells with VP2 exposed, demonstrating that virions needto target the ER to release VP2. We observed that pre-treatment of cellswith Retro-2^(cycl), but not the poorly neutralizing Retro-2^(meta),results in a significant reduction of cells exposing VP2 to similarlevels as those treated with BFA. See FIG. 11C. These punctae colocalizewith the ER rather than other organelles such as lysosomes, see FIG.11B, a result that is consistent with previous studies. See, e.g.,Goodwin et al., mBio (2011) 2:3 e00101-11. Taken together, these datademonstrate that Retro-2^(cycl) treatment of cells reduces ERtrafficking and as a by-product, prevents exposure of the minor capsidproteins of JCPyV, BKPyV, and SV40.

Discussion

We demonstrate that the small molecule Retro-2^(cycl), a recentlydescribed inhibitor of bacterial intoxication, effectively inhibitsinfection by three polyomaviruses. Retro-2^(cycl) inhibits polyomavirusinfection in a similar manner to its effects on ricin and shiga-liketoxins, namely by blocking retrograde trafficking to the ER or Golgi.See, e.g., Stechmann et al., Cell (2010) 141: 231-242. This effectappears specific for viruses that utilize retrograde trafficking, astransduction of cells by an adenovirus pseudovirus is not inhibited byRetro-2^(cycl). This work demonstrates that Retro-2^(cycl) is not onlyan effective antiviral compound, but will also aid to further delineatethe endocytic pathways used by polyomaviruses to target the ER.

During infection, polyomaviruses traffic to the ER where their viralcapsids interact with host cell chaperones to undergo partial uncoatingand utilize the ER associated degradation pathway to gain access to thecytosol. See, e.g., Tsai et al., Curr Top Microbiol Immunol (2010) 343:177-194. We show that Retro-2^(cycl) reduces ER accumulation of virionsand thus prevents this critical step in the infectious lifecycle ofpolyomaviruses. Since virions cannot interact with ER residentchaperones, necessary uncoating steps are inhibited, as can be evidencedby a lack of exposure of the minor capsid protein VP2 in Retro-2^(cycl)treated samples. All polyomaviruses studied to date undergo retrogradetrafficking and in recent years 9 new human polyomaviruses have beendiscovered. See, e.g., Feng et al., Science (2008) 319: 1096-1100;Siebrasse et al., J Virol (2012) 86: 10321-10326; Schowalter et al.,Cell Host Microbe (2010) 7: 509-515; Buck et al., J Virol (2012) 86:10887; van der Meijden et al., PLoS Pathog (2010) 6: e1001024; Allanderet al., J Virol 81: (2007) 4130-4136; Gaynor et al., PLoS Pathog (2007)3: e64; Scuda et al., J Virol (2011) 85: 4586-4590. Several of thesenewly discovered viruses are associated with human diseases, includingMerkel cell polyomavirus, which is the causative agent of the fatalcancer Merkel cell carcinoma. See, e.g., Feng et al., Science (2008)319: 1096-1100. Since Retro-2^(cycl) is protective against JCpyV, BKpyV,and SV40, it is likely that this compound will inhibit replication ofthese polyomaviruses, and will be a useful tool in verifying whetherthese new polyomaviruses target the ER for productive infection.

Brefeldin A is another small molecule that has been described to inhibitER accumulation of polyomaviruses. However, BFA is highly cytotoxic tocells, making this drug less appealing for developments of antiviral oranti-toxin therapies. See, e.g., Barbier et al., Toxins (2012) 4: 15-27.Additionally, BFA treatment rapidly alters the morphology of Golgiapparatus, inhibits endosomal maturation, and inhibits proteinsecretion, demonstrating that this compound elicits numerous off targeteffects besides inhibiting retrograde trafficking. See, e.g.,Lippincott-Schwartz et al., Cell (1989) 56: 801-813; Huotari et al., TheEMBO Journal (2011) 30: 3481-3500; Misumi et al., The Journal ofBiological Bhemistry (1986) 261: 11398-11403; Low et al., The Journal ofBiological Chemistry (1991) 266: 17729-17732. Conversely, Retro-2^(cycl)has been shown to cause little altered cell compartment morphology, andis well tolerated when administered to mice. See, e.g., Stechmann etal., Cell (2010) 141: 231-242. Thus, although Retro-2^(cycl) is not thefirst described inhibitor of ER trafficking of virions, it is likely thefirst small molecule inhibitor of polyomavirus infectivity that showspromise as a potential antiviral therapy.

We also show that the biologically active chemical species of Retro-2 isa dihydroquinazolinone derivative of Retro-2, and not an imine as wasoriginally reported by Stechman and colleagues. See, e.g., Stechmann etal., Cell (2010) 141: 231-242. While completing these studies, anothergroup has confirmed that Retro-2 converts to a dihydroquinazolinone.See, e.g., Park et al., Sci Rep (2012) 2: 631. With the correctstructure of the retrograde transport inhibitor now established, we canconsider the medicinal chemistry optimization of Retro-2^(cycl) as apotential drug lead. An in depth structure activity relationship studyof Retro-2^(cycl) is currently underway in our labs, and we provideherein additional Retro-2^(cycl) analogs synthesized and studied. See,e.g., Table 1, provided below.

The inhibitory effect of Retro-2^(cycl) is strikingly similar to theeffect seen on ricin toxin and shiga-like toxins, where Retro-2^(cycl)treatment prevents endosomal to Golgi trafficking, and as a consequence,also inhibits ER trafficking. See, e.g., Low et al., The Journal ofBiological Chemistry (1991) 266: 17729-17732. This similarity suggestthat there may be overlap in the cellular proteins used by toxins andpolyomaviruses to effect ER trafficking. However, there are likelysignificant differences in the kinetics or pathways used bypolyomaviruses and bacterial toxins to target the ER, since ricin andshiga-like toxins rapidly traffic to the Golgi apparatus, an associationthat has yet to be identified for any polyomavirus. See, e.g., Johanneset al., Nat Rev Microbiol (2010) 8: 105-116. This suggests the cellularhost factors targeted by Retro-2^(cycl) may be involved in multipleretrograde trafficking pathways, only a small proportion of virionstraffic to the Golgi, or that polyomaviruses may transiently trafficthrough the Golgi complex prior to ER accumulation. It is unlikely thatRetro-2^(cycl) is affecting entry steps upstream of ER trafficking, asvirions are still able to bind and enter Retro-2^(cycl) treated cells.Furthermore, Retro-2^(cycl) loses most of its inhibitory effect whenadded to synchronously infected cultures at time points after 14 hours,further demonstrating the binding and entry events are not inhibited byRetro-2^(cycl) treatment. SLTs, CTxB, and some polyomaviruses bind toglycolipids, and may therefore provide some rationale as to how thiscompound is inhibiting trafficking. See, e.g., Ewers et al., Cold SpringHarb Perspect Biol (2011) 3:a004721. However, whereas numerous hostcellular transport factors are known to promote endosomal to Golgitransport of ricin toxins and SLT, such as the retromer complex,Syntaxin 5, EpsinR, and clathrin, the role of these factors inpolyomavirus entry is not known. See, e.g., Popoff et al., J Cell Sci(2007) 120: 2022-2031; Bonifacino et al., Curr Opin Cell Biol (2008) 20:427-436. It is tempting to speculate that these same proteins play acritical role in polyomavirus ER trafficking, and future work examiningthe role of these host factors in polyomavirus entry will increase ourunderstanding of this process. Retro-2^(cycl) treatment causesredistribution of syntaxin 5 and 6 and previous work has demonstratedthat siRNA knockdown of syntaxin 5 significantly inhibits SV40infectivity, suggesting that redistribution of Syntaxin 5 and 6 byRetro-2^(cycl) may inhibit ER trafficking of polyomaviruses. Determiningwhat cellular host factor or factors Retro-2^(cycl) is binding willsignificantly increase our understanding as to how polyomaviruses andtoxins undergo retrograde trafficking.

It is unlikely that Retro-2^(cycl) binds to polyomaviruses directly, andtherefore should decrease the likelihood of escape mutations, sinceinfectious mutants would have to utilize alternate trafficking pathwaysto ultimately deliver their genome to the nucleus for productivereplication. Since the majority of people are persistently infected withJCpyV and BKpyV, the ability of Retro-2^(cycl) to reduce the spread ofJCPyV, BKPyV, and SV40 in established infections suggests that thesecompounds may be efficacious in controlling viral dissemination inpreviously infected individuals. Further optimization of Retro-2^(cycl)should result in effective antiviral therapies to treat or preventdiseases caused by human polyomaviruses or other pathogens that utilizeretrograde trafficking during infection.

Materials and Methods Cells, Viruses, Plasmids, and Antibodies

SVG-A cells were maintained in complete media (minimal essential mediacontaining 10% fetal bovine serum 1% penicillin, 1% streptomycin) andhave been previously described. See Major et al., Proc Natl Acad Sci USA(1985) 82: 1257-1261. Vero cells were purchased from the American TypeCulture Collection (ATCC) and were maintained in complete mediasupplemented with 5% fetal bovine serum. The SVE-delta strain of JCPyV,the Dunlop strain of BKPyV, and the 777 strain of SV40 were used forthese studies, and have been described previously or were purchased fromATCC. See Vacante et al., Virology (1989) 170: 353-361. Alexa Fluor 488labeled cholera toxin was purchased from Invitrogen. Antibodies toprotein disulfide isomerase and VP2 were purchased from Abcam. ThePAB597 and PAB962 hybridoma was a kind gift from Ed Harlow. The mousemonoclonal antibody to SV-40 large T-antigen (AB-2) was purchased fromCalbiochem.

Virus Purification and Labeling

JCPyV, BKPyV, and SV40 were purified similar to previously publishedmethods. See, e.g., Shen et al., Virology (2011) 411: 142-152. Briefly,ten 1700 cm² roller bottles were seeded with SVG-A cells at 50%confluency and infected with JCPyV, BKPyV, or SV40 at an MOI of ˜0.1FFU/cell for 14 days, with the cell culture media replaced at 7 days.Viral lysates were harvested by scraping cells in the presence of cellculture media, and this lysate was frozen and thawed 3 times. The lysatewas then treated with type V neuraminidase (Sigma) at 37° C. for 1 h torelease JCPyV still bound to cells. Deoxycholate acid (FisherScientific) was added to further disrupt cells for 1 h at 37° C. andthen sonicated three times on ice (50% amplitude 50% duty cycle, power4, 1 min). The cellular debris was pelleted by centrifugation, and theviral supernatant was pelleted through a 20% sucrose cushion in aBeckman SW40ti rotor at 150,000×g at 4° C. for 3 h. The viral pellet wasresuspended into buffer A (10 mM Tris-HCl, 50 mM NaCl, 0.1 mM CaCl₂) andsonicated 3 times (30% amplitude 50% duty cycle, power 3, 1 min). Theresuspended pellet was loaded onto a CsCl step gradient (1.29-1.35 g/ml)and spun at 115,000×g at 4° C. for 18 h in a Beckman SW55ti rotor. Theband corresponding to DNA-containing virions was isolated and dialyzedextensively against buffer A. JCPyV, BKPyV, or SV40 was labeled withAlexa Fluor 488 or Alexa Fluor 633 according to the manufacturer'sinstructions (Invitrogen). Briefly, purified JCPyV was dialyzed in 0.1Mcarbonate/bicarbonate buffer pH 8 and the virus was then concentrated to˜0.5 mg/ml using a 10,000 MWCO Amicon Ultra (Millipore) and dye wasadded at a molar excess of 200:1. After 1 h of rocking incubation atroom temperature, the excess dye was removed by extensive dialysisagainst buffer A.

Dose Dependent Inhibition of Infection by Retro-2^(cycl)

SVG-A or Vero cells were seeded into 12-well plates at a concentrationof 2×10⁵ cells per well. The next day cells were pre-treated withRetro-2^(cycl) for 0.5 h, then virus was bound to cells in 0.08 mL inthe presence of Retro compounds at the indicated concentration. Afterallowing adsorption of virions to cells for 1 h, 2 mL per well ofcomplete media containing Retro-2 at the indicated concentration wasoverlaid into each well. The final concentration of DMSO in all wellswas 0.04%. After allowing cells to be infected for 48 h to allowexpression of large T-antigen or 72 h for VP1 expression, infected cellswere scored by flow cytometry. Curve fitting was performed using Origin(OriginLab), with the non-linear curve fitting package (Growth/Sigmodialcategory, Dose response function), with shared and fixed parameters of 0and 100. EC₅₀ values were calculated for each replicate and these valueswere used to generate average EC₅₀ data.

Flow Cytometric Scoring of Viral Infection

SVG-A or Vero cells were detached from 12-well plates by aspirating thegrowth media, washing adherent cells once with phosphate buffered saline(PBS), and detaching with an EDTA based non-enzymatic agent(Cellstripper). These cells were then transferred to v-bottom 96-wellplates and pelleted by centrifugation at 600×g for 5 min, washed withPBS and fixed in 0.2 mL 4% paraformaldahyde (PFA) for 10 min. Cells werepelleted and washed with PBS, and permeabalized with 0.2 mL PBScontaining 1% Triton X-100 for 10 min at 21° C. Cells were then pelletedand resuspended in 0.1 mL PBS containing 3% BSA and an Alexa Fluorlabeled monoclonal antibody to VP1 (PAB 597-AF488) or a polyclonalantibody to large T-antigen (AB-2 for BKPyV and SV40 or JCT962 forJCPyV). After incubation for 1 h at 21° C., cells were washed once withPBS and fluorescence was read by flow cytometry on a (FACSCanto II, BDBiosciences). For large T-antigen staining, cells were washed 3 timesand then stain with a secondary antibody conjugated to Alexa fluor 488for 1 h at room temp. Uninfected cells were used to establish gates forinfected cells. An infected culture without Retro compounds (˜10% totalinfected cells) was then normalized to 100% and any reduction ininfection in the Retro-2^(cycl) treated cells were then compared to thisuntreated control. Three independent experiments were performed and usedto calculate standard deviations. T-tests were used to calculatedsignificance.

Time Course Experiments

SVG-A or Vero cells were seeded into 12-well plates in 1 mL completemedia at a concentration of 2×10⁵ cells per well. The following day,cells were chilled to 4° C. to prevent endocytosis, and virions werebound for 1 h at 4° C. SVG-A cells were infected with JCPyV, and Verocells were infected with BKPyV, or SV40 at an multiplicity of infection(MOI) of 1. After binding, excess virus was washed away with chilledmedia, and samples were warmed to 37° C. At each indicated time point,media was removed, and fresh media containing Retro compounds was addedto each sample. After 72 h, cells were scored for infection by flowcytometry.

Multicycle Growth Assays

Replicate 24-well plates were seeded with SVG-A or Vero cells at 25%confluency. The next day cells were infected with the appropriate virusat an MOI of 0.01 in 0.04 mL of complete media for 1 h at 37° C. Afterthis incubation, 2 mL of compete media was then added to each well for 3days to allow infection to be established. After 72 h, 100 μM of Retro-2was added to each appropriate replicates. This media was aspirated andreplace with fresh media containing Retro-2 daily. Replicates of eachsample was fixed every 3 days and stained for VP1. At each day, thereplicates containing Retro compounds were compared to the untreatedcontrols. For the reinfection assays, the supernatants were saved andused to reinfect naïve cells. After 72 h, infected cells were thenscored by flow cytometry.

Microscopy

All microscopy experiments were performed using an LSM-710 laserscanning confocal microscope with a 63× 1.4 NA plan apochromat objectivewith the pinhole set to one Airy unit (Carl Zeiss). DAPI was excitedusing a 405 nm diode laser, Alexa Fluor 488 was excited using a 488 nmargon laser and Alexa Fluor 633 was excited using a 633 nm helium-neonlaser. For the endocytosis assay, SVG-A or Vero cells were seeded in#1.5 glass-bottomed 96-well plates at a density of 3×10³ cells and wereincubated overnight. The following day, cells were pretreated with 100μM Retro-2, 0.04% DMSO, 30 μM chloropromazine, or 100 μM nystatin for 30min at 37° C. Cells were then chilled to 4° C. to prevent endocytosis,and AF488-JCPyV, BKpyV, or SV40 was added at an MOI of 1 for 1 h withrocking. Unbound virus was removed by washing with chilled medium and 1mL of media containing the appropriate drug was then added to each welland samples were returned to the incubator for 2 h at 37° C. prior tofixation with 4% PFA. Confocal Z-series were acquired, and trypan bluewas added at a final concentration of 0.008% to quench the fluorescenceon non-internalized virions. After quenching, the same cell wasreimaged. Fluorescence from virions prior to quenching was pseudocoloredgreen and post quenching fluorescence was pseudocolored red. Image noisewas reduced using a 3:3:3 median filter and maximal image projectionsgenerated. Images were aligned and brightness and contrast was adjustedusing Adobe Photoshop. For the ER trafficking assay, cells were platedand infected similar to the endocytosis assay, with cells inoculatedwith Alexa fluor 488 labeled polyomaviruses at an MOI of 1. At 8 hpi (3hpi for CTxB), cells were fixed and the endoplasmic reticulum wasstained with a polyclonal antibody to PDI and visualized with an Alexafluor 633 labeled secondary antibody. Image noise was reduced using a3:3:3 median filter and samples were bandpassed using an FFT filter inImageJ to enhance edges, as has previously been reported (settings: 15pixels large structures, 3 pixels small structures, 5% tolerance).Colocalization was determined by inoculating cells with unlabeled JCpyV,BKpyV, or SV40 at an MOI of 1 or Alexa fluor 488 labeled CTxB (4 gg/ml)for 1 h, samples were wash with media containing drug, and fixed at 8hpi. Virions were immunostained with a mouse monocolonal antibody to VP1(PAb597) and PDI was stained with a rabbit polyclonal antibody to PDI.Colocalization was determined using Manders coefficient ofcolocalization, using the JACoP plugin for ImageJ. M1 values werenormalized to a DMSO control and 5 cells were imaged for each conditiontested.

Significance Testing was Performed Using T-Test

For the VP2 release assay, cells were seeded 12-well plates in 1 mLcomplete media at a concentration of 5×10⁴ cells per well and incubatedovernight. The following day, cells were pretreated with 100 μM Retro-2,0.04% DMSO, or 500 ng/mL of brefeldin A for 30 min. Samples were theninoculated with JCpyV, BKpyV, or SV40 at an MOI of 10 for 1 h withrocking at 37° C. Excess virus was washed off and samples were incubatedin the presence of inhibitors and fixed at 10 hpi. These cells were thenstained with a polyclonal antibody to VP2 and counter-stained with DAPI.At least ten independent fields of view were counted per sample, andthree independent replicates were performed, with an average of 1700cells counted per condition. Significance testing was performed usingT-test. To visualize colocalization between VP2 and the ER, Vero cellswere infected with SV40 as for the VP2 release assay. After fixation,cells were stained for VP1 using the mouse monoclonal antibody PAB597,VP2 was stained using a rabbit polyclonal antibody, and the ER wasstained using a goat polyclonal antibody to PDI. The nucleus wascounter-stained with BOBO-3. Image noise was reduced using a 3:3:3median filter, and a single Z-slice was shown.

Synthetic Procedures

Retro-2 compound was either purchased from Chembridge, or weresynthesized in house. Stock solutions were generated by dissolvingRetro-2 in DMSO. When added to cells, the final concentration of DMSOused in these studies was 0.04% (v/v). Additional compounds weresynthesized following general synthetic methods described herein. Allcommercially available reagents were used without further purification.Reactions were carried out in oven dried glassware, with dry solvent,and under ambient atmosphere. All spectra were referenced to residualsolvent signals in DMSO_(d6) (2.50 ppm for ¹H, 39.51 ppm for ¹³C).

Dihydroquinazolinone Synthesis; General Procedure 1

Dicyclohecylcarbodiimide (5.00 mmol) and a nitrobenzoic acid (5.55 mmol)were dissolved in DCM (20 mL) and allowed to stir for 5 minutes beforethe addition of a primary amine (5.55 mmol) and dimethylaminopyridine(0.055 mmol). The coupling reaction was allowed to proceed for 16 hours,after which the DCM was evaporated. The solid residue was resuspended inethyl acetate and filtered through a silica gel plug to remove thedicyclohexylurea byproduct. The filtrate was then concentrated and thedesired nitrobenzamide isolated by silica gel chromatography using ahexanes/ethyl acetate solvent gradient.

The nitrobenzamide intermediate (0.5 mmol), an aldehyde (0.55 mmol) andscandium (III) triflate (0.05) mmol were combined in methanol (1 mL) ina sealed microwave vial. The reaction was microwave irradiated at 100°C. for 1 hour, after which the solvent was removed. The productdihydroquinazolinones were isolated by silica gel chromatography with ahexanes/ethyl acetate solvent gradient. The chromatographed productswere subsequently purified by recrystallization.

Dihydroquinazolinone Synthesis; General Procedure 2

An isatoic anhydride (1.20 mmol) was added to THF (6 mL) and heated to60° C. To the hot solution of isatoic anhydride was added a primaryamine or ammonia (1.00 mmol), which was allowed to react for 1-2 hours.Once the amine had been completely consumed, an aldehyde (1.20 mmol) andscandium triflate (0.1 mmol) were added and allowed to react at 60° C.for an additional 3-5 hours, after which the solvent was removed. Theproduct dihydroquinazolinones were isolated by silica gel chromatographywith a hexanes/ethyl acetate solvent gradient. The chromatographedproducts were subsequently purified by recrystallization.

Synthesis of Retro-2 and Retro-2^(cycl)

To a stirring solution of 2-aminobenzanilide was added5-methyl-2-thiophenecarboxaldehyde (86.3 μL, 0.80 mmol). After 24 hours,the reaction was concentrated and then chromatographed on silica gelwith 20-40% ethyl acetate in hexanes. Retro-2 and Retro-2^(cycl) werecleanly separated and then further purified by recrystallization fromethanol and ethyl acetate respectively. Yields: Retro-2 47.2 mg (36%),Retro-2^(cycl) 78.0 mg (59.4%). Characterization: Retro-2 FAB HRMS:C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881 Found: 343.0870. Retro-2^(cycl) FABHRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881 Found: 343.0888.

Synthesis of Retro-2^(Red)

Retro-2^(cycl) (143 mg, 0.444 mmol) was dissolved in methanol (4 mL)then treated with sodium cyanoborohydride (83 mg, 1.3 mmol) followed byacetic acid (0.4 mL). The slow formation of a new product was observedby tlc (2:1 hexanes:ethyl acetate). After 3 days the reaction wasconcentrated and then chromatographed on silica gel with a hexanes/ethylacetate solvent gradient. Both the desired product (62 mg, 0.187 mmol,42%) as well as unreacted starting material (64 mg, 0.200 mmol, 45%)were recovered. FAB HRMS: C₁₉H₁₈N₂OSNa⁺ Predicted: 345.1038 Found:345.1052.

Synthesis of Retro-2^(meta)

Dicyclohecylcarbodiimide (1.03 g, 5.00 mmol) and 3-nitrobenzoic acid(0.987 g, 5.55 mmol) were dissolved in DCM (20 mL) and allowed to stirfor 5 minutes before the addition of aniline (0.500 mL, 5.55 mmol) anddimethylaminopyridine (0.068 g, 0.055 mmol). The coupling reaction wasallowed to proceed for 16 hours at which point the DCM was evaporated.The solid residue was resuspended in ethyl acetate and filtered througha silica gel plug to remove the dicyclohexylurea byproduct. The filtratewas then concentrated and the desired nitrobenzamide isolated by silicagel chromatography using a hexanes/ethyl acetate solvent gradient.Yield: 1.118 g, 92%. FAB HRMS: C₁₃H₁₀N₂O₃Na⁺ Predicted: 265.0589 Found:265.0595. 1H NMR (600 MHz, DMSO-d6) δ=10.57 (s, 1H), 8.79 (t, J=1.8 Hz,1H), 8.44 (ddd, J=0.9, 2.3, 8.2 Hz, 1H), 8.41 (td, J=1.3, 7.7 Hz, 1H),7.84 (t, J=8.1 Hz, 1H), 7.79 (d, J=7.7 Hz, 2H), 7.39 (d, J=7.7 Hz, 1H),7.38 (d, J=7.7 Hz, 1H), 7.14 (tt, J=1.1, 7.3 Hz, 1H) 13C NMR (151 MHz,DMSO-d6) δ=163.3, 147.7, 138.6, 136.3, 134.1, 130.1, 128.6, 126.1,124.1, 122.4, 120.6.

The Nitrobenzamide (0.242 g, 1.00 mmol) was dissolved in methanol (3 mL)then treated with 10% Paladium on carbon (72.3 mg) and ammonium formate(350 mg, 5.5 mmol). The reaction was allowed to proceed for 45 minutesbefore being filtered through celite to remove the palladium on carbon.The filtrate was treated with 5-methyl-2-thiophenecarboxaldehyde (0.128mL, 1.2 mmol) and scandium (III) triflate (0.076 g, 0.154 mmol) and thenheated to reflux for 2 hours. Upon cooling to room temperature, theproduct precipitated from solution and was isolated by filtration.yield: 0.190 g, 59%. FAB HRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881 Found:343.0888.

TABLE 1 Structure Name Characterization data

Retro-2 FAB HRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881 Found: 343.0870. ¹HNMR (600 MHz, DMSO-d₆) δ = 11.00 (s, 1 H), 8.79 (s, 1 H), 8.00 (dd, J =1.5, 7.7 Hz, 1 H), 7.76 (d, J = 7.7 Hz, 2 H), 7.65 (d, J = 3.7 Hz, 1 H),7.59 (dt, J = 1.5, 7.7 Hz, 1 H), 7.42-7.32 (m, 4 H), 7.10 (s, 1 H), 7.01(dd, J = 1.1, 3.7 Hz, 1 H), 2.55 (s, 3 H). ¹³C NMR (151 MHz, DMSO-d₆) δ= 164.1, 155.7, 148.4, 147.4, 139.3, 138.6, 136.2, 132.3, 129.9, 128.8,128.0, 127.5, 126.2, 123.6, 119.8, 119.4, 15.7

RETRO-2cycl Retro-2^(cycl) FAB HRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881Found: 343.0888 ¹H NMR (400 MHz, DMSO-d₆) δ = 7.73 (d, J = 8.0 Hz, 1 H),7.61 (d, J = 2.8 Hz, 1 H), 7.41-7.34 (m, 2 H), 7.34-7.28 (m, 3 H),7.27-7.20 (m, 1 H), 6.82 (d, J = 8.3 Hz, 1 H), 6.77 (t, J = 7.5 Hz, 1H), 6.72 (d, J = 3.5 Hz, 1 H), 6.55 (d, J = 3.5 Hz, 1 H), 6.40 (d, J =3.0 Hz, 1 H), 2.31 (s, 3 H). ¹³C NMR (151 MHz, DMSO-d₆) δ = 161.5,146.3, 142.0, 140.5, 139.2, 133.7, 128.7, 127.9, 126.4, 126.3, 126.2,124.4, 117.9, 115.5, 115.2, 69.6, 14.9

BU62382A-red Retro-2red FAB HRMS: C₁₉H₁₈N₂OSNa⁺ Predicted: 345.1038Found: 345.1052 ¹H NMR (600 MHz, DMSO-d₆) δ = 10.11 (s, 1 H), 7.78 (t, J= 5.7 Hz, 1 H), 7.76-7.65 (m, 3 H), 7.45-7.28 (m, 3 H), 7.16-7.06 (m, 1H), 6.85 (d, J = 2.9 Hz, 1 H), 6.81 (d, J = 8.4 Hz, 1 H), 6.68 (t, J =7.5 Hz, 1 H), 6.66- 6.61 (m, 1 H), 2.38 (s, 3 H). ¹³C NMR (151 MHz,DMSO-d₆) δ = 168.0, 148.5, 140.5, 139.0, 138.1, 132.6, 129.0, 128.5,125.0, 124.9, 123.5, 120.6, 116.1, 114.9, 111.6, 41.6, 14.9.

BU62382A-meta Retro-2meta FAB HRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881Found: 343.0888. ¹H NMR (600 MHz, DMSO-d₆) δ = 10.28 (s, 1 H), 8.78 (s,1 H), 7.82 (t, J = 1.7 Hz, 1 H), 7.82- 7.78 (m, 3 H), 7.56-7.52 (m, 2H), 7.45 (ddd, J = 1.1, 2.0, 7.9 Hz, 1 H), 7.38-7.33 (m, 2 H), 7.11 (tt,J = 1.1, 7.3 Hz, 1 H), 6.97-6.94 (m, 1 H), 2.53 (d, J = 0.7 Hz, 3 H).¹³C NMR (151 MHz, DMSO-d₆) δ = 165.1, 154.7, 150.9, 145.9, 140.1, 139.1,135.9, 134.6, 129.3, 128.5, 126.9, 125.1, 124.0, 123.6, 120.4, 120.2,15.6

BU62382A1 RETRO2A1 FAB HRMS: C₁₈H₁₄N₂OSNa⁺ Predicted: 329.0725 Found:329.0735. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.72 (dd, J = 1.8, 7.8 Hz, 1 H),7.62 (d, J = 3.3 Hz, 1 H), 7.60-7.56 (m, 1 H), 7.42-7.35 (m, 2 H),7.34-7.28 (m, 3 H), 7.28-7.23 (m, 1 H), 6.82 (dd, J = 0.8, 8.3 Hz, 1 H),6.75 (s, 1 H), 6.33 (dd, J = 1.8, 3.3 Hz, 1 H), 6.24 (t, J = 3.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ = 161.6, 146.3, 144.6, 140.5, 133.8,128.7, 128.0, 126.5, 126.4, 126.4, 126.3, 125.9, 118.1, 115.6, 115.3,69.5

BU62382A2 RETRO2A2 FAB HRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881 Found:343.0872. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.73 (dd, J = 1.5, 7.8 Hz, 1 H),7.62 (d, J = 2.8 Hz, 1 H), 7.42-7.29 (m, 5 H), 7.27-7.22 (m, 1 H),6.94-6.90 (m, 1 H), 6.82 (d, J = 8.0 Hz, 1 H), 6.80-6.74 (m, 2 H), 6.44(d, J = 2.8 Hz, 1 H), 2.07 (d, J = 0.8 Hz, 3 H). ¹³C NMR (101 MHz,DMSO-d₆) δ = 161.5, 146.3, 144.6, 140.5, 136.2, 133.8, 128.7, 128.3,128.0, 126.3, 126.3, 120.9, 118.0, 115.6, 115.3, 69.4, 15.3.

BU62382A3 RETRO2A3 FAB HRMS: C₁₉H₁₆N₂OSNa⁺ Predicted: 343.0881 Found:343.0885. ¹H NMR (400 MHz, DMSO-d₆) δ =7.74 (dd, J = 1.5, 7.8 Hz, 1 H),7.48 (d, J = 2.0 Hz, 1 H), 7.37-7.29 (m, 3 H), 7.25-7.17 (m, 4 H),6.83-6.75 (m, 2 H), 6.67 (d, J = 5.0 Hz, 1 H), 6.54 (d, J = 2.0 Hz, 1H), 1.91 (s, 3 H). ¹³C NMR (75 MHz, DMSO-d₆) δ = 161.8, 146.6, 140.2,137.0, 135.1, 133.8, 129.5, 128.6, 127.9, 127.5, 126.7, 124.2, 117.8,115.0, 114.9, 67.9, 13.3.

BU62382A4 RETRO2A4 FAB HRMS: C₂₀H₁₈N₂OSNa⁺ Predicted: 357.1038 Found:357.1028. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.74 (dd, J = 1.5, 7.8 Hz, 1 H),7.63 (d, J = 2.8 Hz, 1 H), 7.41-7.34 (m, 2 H), 7.34-7.29 (m, 3 H),7.27-7.21 (m, 1 H), 6.83 (d, J = 8.0 Hz, 1 H), 6.80-6.76 (m, 1 H), 6.75(d, J = 3.5 Hz, 1 H), 6.58 (td, J = 1.1, 3.3 Hz, 1 H), 6.42 (d, J = 2.8Hz, 1 H), 2.67 (q, J = 7.4 Hz, 2 H), 1.13 (t, J = 7.4 Hz, 3 H). ¹³C NMR(75 MHz, DMSO-d₆) δ = 161.5, 146.7, 146.3, 141.7, 140.5, 133.8, 128.7,128.0, 126.4, 126.3, 126.1, 122.7, 118.0, 115.5, 115.2, 69.7, 22.7, 15.6

BU62382A5 RETRO2A5 FAB HRMS: C₂₂H₁₆N₂OSNa⁺ Predicted: 379.0881 Found:379.0866. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.88-7.82 (m, 1 H), 7.79-7.71(m, 3 H), 7.42-7.37 (m, 4 H), 7.37-7.33 (m, 1 H), 7.33-7.28 (m, 3 H),7.28-7.22 (m, 1 H), 6.86 (d, J = 8.0 Hz, 1 H), 6.80 (dt, J = 1.0, 7.5Hz, 1 H), 6.64 (d, J = 3.3 Hz, 1 H). ¹³C NMR (101 MHz, DMSO-d₆) δ =161.4, 146.2, 145.3, 144.8, 140.4, 138.3, 134.0, 128.8, 128.0, 126.5,124.9, 124.5, 123.8, 123.0, 122.6, 118.2, 115.6, 115.3, 107.6, 69.9.

BU62382A6 RETRO2A6 FAB HRMS: C₁₈H₁₄N₂O₂Na⁺ Predicted: 313.0953 Found:313.0945. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.72 (d, J = 7.3 Hz, 1 H), 7.62(d, J = 3.0 Hz, 1 H), 7.58 (dd, J = 0.8, 1.9 Hz, 1 H), 7.43-7.35 (m, 2H), 7.35-7.28 (m, 3 H), 7.28-7.23 (m, 1 H), 6.83 (d, J = 8.0 Hz, 1 H),6.79-6.70 (m, J = 1.0, 7.5, 7.5 Hz, 1 H), 6.33 (dd, J = 1.9, 3.4 Hz, 1H), 6.25 (t, J = 3.3 Hz, 2 H). ¹³C NMR (75 MHz, DMSO-d₆) δ = 161.8,152.9, 146.5, 143.1, 140.6, 133.7, 128.7, 127.9, 126.3, 117.8, 115.5,114.9, 110.4, 108.4, 67.3

BU62382A7 RETRO2A7 FAB HRMS: C₁₉H₁₆N₂O₂Na⁺ Predicted: 327.1109 Found:327.1125. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.72 (dd, J = 1.0, 7.8 Hz, 1 H),7.62 (d, J = 3.0 Hz, 1 H), 7.42-7.33 (m, 4 H), 7.30 (ddd, J = 1.8, 7.0,8.3 Hz, 1 H), 7.24 (tt, J = 1.8, 7.3 Hz, 1 H), 6.83 (d, J = 8.0 Hz, 1H), 6.78-6.72 (m, 1 H), 6.17 (d, J = 3.3 Hz, 1 H), 6.11 (d, J = 3.0 Hz,1 H), 5.93 (dd, J = 1.0, 3.0 Hz, 1 H), 2.16 (s, 3 H). ¹³C NMR (75 MHz,DMSO-d₆) δ = 161.8, 151.7, 150.8, 146.4, 140.6, 133.6, 128.7, 127.9,126.3, 117.7, 115.5, 115.0, 109.4, 106.5, 67.3, 13.3.

BU62382A8 RETRO2A8 FAB HRMS: C₁₈H₁₅N₃ONa⁺ Predicted: 312.1113 Found:312.1118. ¹H NMR (400 MHz, DMSO-d₆) δ = 10.72 (br. s., 1 H), 7.72 (dd, J= 1.5, 7.8 Hz, 1 H), 7.34- 7.26 (m, 3 H), 7.26-7.21 (m, 3 H), 7.21- 7.15(m, 1 H), 6.79 (dd, J = 0.5, 8.3 Hz, 1 H), 6.75 (ddd, J = 1.0, 7.1, 7.5Hz, 1 H), 6.63 (dt, J = 1.5, 2.6 Hz, 1 H), 6.18 (d, J = 2.3 Hz, 1 H),5.89 (t, J = 3.5 Hz, 1 H), 5.81 (q, J = 2.7 Hz, 1 H). ¹³C NMR (75 MHz,DMSO-d₆) δ = 162.4, 146.8, 140.7, 133.4, 130.0, 128.5, 127.9, 126.6,126.1, 118.2, 117.8, 115.9, 115.1, 107.6, 107.1, 68.1.

BU62382B1 RETRO2A4B1 FAB HRMS: C₂₁H₂₀N₂OSNa⁺ Predicted: 371.1194 Found:371.1186. ¹H NMR (600 MHz, DMSO-d₆) δ = 7.70 (dd, J = 1.8, 7.7 Hz, 1 H),7.37 (d, J = 2.6 Hz, 1 H), 7.36-7.33 (m, 2 H), 7.32-7.30 (m, 2 H),7.30-7.25 (m, 2 H), 6.87 (d, J = 3.7 Hz, 1 H), 6.74 (dt, J = 1.1, 7.5Hz, 1 H), 6.71 (d, J = 8.1 Hz, 1 H), 6.65 (td, J = 1.1, 3.3 Hz, 1 H),5.90 (d, J = 2.6 Hz, 1 H), 5.26 (d, J = 15.4 Hz, 1 H), 3.92 (d, J = 15.4Hz, 1 H), 2.69 (dq, J = 1.1, 7.5 Hz, 2 H), 1.14 (t, J = 7.5 Hz, 3 H).¹³C NMR (151 MHz, DMSO- d₆) δ = 161.7, 146.6, 146.1, 141.2, 137.5,133.4, 128.4, 127.6, 127.4, 127.1, 126.0, 122.6, 117.7, 114.8, 114.7,66.5, 46.7, 22.7, 15.6.

BU62382B2 RETRO2A4B2 FAB HRMS: C₁₈H₂₂N₂OSNa⁺ Predicted: 337.1351 Found:337.1366. ¹H NMR (600 MHz, DMSO-d₆) δ = 7.64 (dd, J = 1.5, 8.1 Hz, 1 H),7.33 (d, J = 2.6 Hz, 1 H), 7.27-7.21 (m, 1 H), 6.88 (d, J = 3.7 Hz, 1H), 6.73-6.67 (m, 2 H), 6.64 (td, J = 1.0, 3.6 Hz, 1 H), 6.00 (d, J =2.6 Hz, 1 H), 3.86 (ddd, J = 6.6, 8.4, 13.6 Hz, 1 H), 2.83 (ddd, J =5.5, 8.3, 13.7 Hz, 1 H), 2.68 (dq, 1.1, 7.5 Hz, 2 H), 1.59 1.51 (m, 1H), 1.51-1.43 (m, 1 H), 1.34-1.21 (m, 2 H), 1.13 (t, J = 7.5 Hz, 3 H),0.87 (t, J = 7.3 Hz, 3 H). ¹³C NMR (151 MHz, DMSO-d₆) δ = 161.5, 146.4,146.0, 142.0, 133.1, 127.4, 125.7, 122.5, 117.5, 115.2, 114.6, 66.7,43.7, 29.5, 22.6, 19.5, 15.6, 13.6.

BU62382B3 RETRO2A4B3 FAB HRMS: C₁₇H₂₀N₂OSNa⁺ Predicted: 323.1194 Found:323.1188. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.68-7.61 (m, 1 H), 7.36 (br.s., 1 H), 7.24 (dt, J = 1.5, 7.7 Hz, 1 H), 6.88 (d, J = 3.5 Hz, 1 H),6.74- 6.67 (m, 2 H), 6.63 (d, J = 3.5 Hz, 1 H), 6.01 (d, J = 2.3 Hz, 1H), 3.88-3.74 (m, 1 H), 2.81 (ddd, J = 5.7, 8.1, 13.5 Hz, 1 H), 2.67 (q,J = 7.6 Hz, 2 H), 1.67-1.55 (m, 1 H), 1.55-1.42 (m, 1 H), 1.13 (t, J =7.6 Hz, 3 H), 0.85 (t, J = 7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ =161.6, 146.4, 146.1, 142.1, 133.2, 127.4, 125.7, 122.6, 117.5, 115.2,114.7, 66.8, 45.8, 22.7, 20.8, 15.7, 11.2.

BU62382B5 RETRO2A4B5 FAB HRMS: C₂₀H₂₄N₂OSNa⁺ Predicted: 363.1507 Found:363.1522. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.65 (dd, J = 1.2, 7.7 Hz, 1 H),7.28 (d, J = 2.4 Hz, 1 H), 7.21 (ddd, J = 1.5, 6.8, 8.6 Hz, 1 H), 6.85(d, J = 3.7 Hz, 1 H), 6.70 (t, J = 7.5 Hz, 1 H), 6.66 (d, J = 8.1 Hz,163 H), 6.58 (d, J = 3.4 Hz, 1 H), 6.08 (d, J = 2.9 Hz, 1 H), 4.23 (t, J= 11.9 Hz, 1 H), 2.64 (q, J = 7.4 Hz, 2 H), 1.76 (d, J = 10.8 Hz, 2 H),1.68 (br. s., 1 H), 1.57 (br. s., 3 H), 1.39-1.15 (m, 4 H), 1.11 (t, J =7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.2, 145.7, 145.7, 144.0,133.0, 127.6, 125.2, 122.4, 117.6, 116.4, 114.9, 63.0, 53.4, 30.3, 30.3,25.7, 25.6, 24.9, 22.6, 15.5.

BU62382B6 RETRO2A4B6 FAB HRMS: C₁₇H₂₀N₂OSNa⁺ Predicted: 323.1194 Found:323.1180. ¹H NMR (600 MHz, DMSO-d₆) δ = 7.66 (dd, J = 1.1, 7.7 Hz, 1 H),7.26 (d, J = 2.9 Hz, 1 H), 7.22 (ddd, J = 1.5, 7.3, 8.2 Hz, 1 H), 6.87(d, J = 3.7 Hz, 1 H), 6.71 (t, J = 7.5 Hz, 1 H), 6.68 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 3.7 Hz, 1 H), 6.06 (d, J = 2.9 Hz, 1 H), 4.55 (spt, J =6.8 Hz, 1 H), 2.64 (q, J = 7.5 Hz, 2 H), 1.23 (d, J = 7.0 Hz, 3 H), 1.11(t, J = 7.5 Hz, 3 H), 1.04 (d, J = 7.0 Hz, 3 H). ¹³C NMR (151 MHz,DMSO-d₆) δ = 161.2, 145.8, 145.7, 143.8, 132.9, 127.5, 125.2, 122.3,117.6, 116.3, 114.8, 63.0, 45.6, 22.6, 20.3, 20.1, 15.5.

BU62382B8 RETRO2A4B8 FAB HRMS: C₂₅H₂₂N₂OSNa⁺ Predicted: 421.1351 Found:421.1359. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.13-8.06 (m, 1 H), 8.00-7.94(m, 1 H), 7.91 (d, J = 7.8 Hz, 1 H), 7.76 (d, J = 7.8 Hz, 1 H), 7.58-7.46 (m, 4 H), 7.34 (d, J = 2.3 Hz, 1 H), 7.32-7.26 (m, 1 H), 6.91 (d, J= 3.5 Hz, 1 H), 6.80-6.74 (m, 1 H), 6.68 (d, J = 8.3 Hz, 1 H), 6.67-6.64(m, 1 H), 5.89 (d, J = 15.4 Hz, 1 H), 5.80 (d, J = 2.5 Hz, 1 H), 4.22(d, J = 15.7 Hz, 1 H), 2.69 (q, J = 7.5 Hz, 2 H), 1.14 (dt, J = 1.3, 7.4Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.6, 146.6, 146.0, 140.8,133.7, 133.5, 132.2, 131.1, 128.6, 128.2, 127.8, 126.5, 126.2, 126.0,125.5, 123.6, 122.7, 117.8, 114.9, 114.7, 65.8, 44.4, 22.7, 15.7.

BU62382B10 RETRO2A4B10 FAB HRMS: C₁₄H₁₄N₂OSNa⁺ Predicted: 281.0725Found: 287.0730. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.39 (br. s, 1 H), 7.60(dd, J = 1.5, 7.8 Hz, 1 H), 7.28-7.22 (m, 1 H), 7.21 (br. s, 1 H), 6.91(d, J = 3.3 Hz, 1 H), 6.74 (d, J = 8.1 Hz, 1 H), 6.72- 6.66 (m, 2 H),5.92 (t, J = 1.8 Hz, 1 H), 2.74 (dq, J = 0.8, 7.6 Hz, 2 H), 1.18 (t, J =7.6 Hz, 3 H). ¹³C NMR (151 MHz, DMSO- d₆) δ = 163.0, 147.2, 146.7,143.4, 133.3, 127.2, 125.3, 122.7, 117.4, 115.0, 114.6, 62.8, 22.8,15.8.

BU62382B11 RETRO2A4B11 FAB HRMS: C₂₂H₂₂N₂O₂SNa⁺ Predicted: 401.1300Found: 401.1285. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.69 (dd, J = 1.3, 7.6Hz, 1 H), 7.34 (d, J = 2.8 Hz, 1 H), 7.32-7.19 (m, 3 H), 6.90 (d, J =8.6 Hz, 2 H), 6.86 (d, J = 3.5 Hz, 1 H), 6.73 (t, J = 7.5 Hz, 1 H), 6.69(d, J = 7.8 Hz, 1 H), 6.65 (d, J = 3.5 Hz, 1 H), 5.84 (d, J = 2.5 Hz, 1H), 5.22 (d, J = 14.9 Hz, 1 H), 3.80 (d, J = 14.9 Hz, 1 H), 3.73 (s, 3H), 2.68 (q, J = 7.5 Hz, 2 H), 1.14 (t, J = 7.6 Hz, 3 H). ¹³C NMR (101MHz, DMSO-d₆) δ = 161.6, 158.6, 146.6, 146.1, 141.3, 133.5, 129.3,129.1, 127.6, 126.0, 122.7, 117.7, 114.8, 114.7, 113.9, 66.2, 55.1,46.0, 22.7, 15.7.

BU62382B12 RETRO2A4B12 FAB HRMS: C₂₂H₂₂N₂O₂SNa⁺ Predicted: 401.1300Found: 401.1282. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.69 (dd, J = 1.4, 7.7Hz, 1 H), 7.39 (d, J = 2.5 Hz, 1 H), 7.32-7.22 (m, 2 H), 6.92-6.82 (m, 4H), 6.78-6.69 (m, 2 H), 6.64 (d, J = 3.3 Hz, 1 H), 5.90 (d, J = 2.8 Hz,1 H), 5.23 (d, J = 15.4 Hz, 1 H), 3.90 (d, J = 15.4 Hz, 1 H), 3.72 (s, 3H), 2.68 (q, J = 7.5 Hz, 2 H), 1.14 (t, J = 7.6 Hz, 3 H). ¹³C NMR (101MHz, DMSO-d₆) δ = 165.2, 159.4, 146.6, 141.3, 139.1, 133.5, 129.6,127.6, 126.0, 122.6, 119.6, 117.7, 114.8, 113.2, 112.4, 66.6, 55.0,46.7, 22.7, 15.7.

BU62382B13 RETRO2A4B13 FAB HRMS: C₂₂H₂₂N₂O₂SNa⁺ Predicted: 401.1300Found: 401.1289. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.67 (dd, J = 1.4, 7.7Hz, 1 H), 7.41 (d, J = 2.8 Hz, 1 H), 7.32-7.24 (m, 2 H), 7.22 (d, J =7.3 Hz, 1 H), 7.02 (d, J = 7.8 Hz, 1 H), 6.93 (t, J = 7.5 Hz, 1 H), 6.85(d, J = 3.5 Hz, 1 H), 6.77-6.69 (m, 2 H), 6.67-6.63 (m, 1 H), 5.92 (d, J= 2.3 Hz, 1 H), 5.11 (d, J = 15.9 Hz, 1 H), 3.96 (d, J = 15.9 Hz, 1 H),3.81 (s, 3 H), 2.69 (q, J = 7.6 Hz, 2 H), 1.14 (t, J = 7.5 Hz, 3 H). ¹³CNMR (101 MHz, DMSO-d₆) δ = 157.0, 146.2, 141.5, 133.5, 128.4, 127.7,127.6, 125.9, 122.7, 120.4, 117.7, 114.9, 114.8, 110.7, 66.8, 55.4,42.1, 22.7, 15.7.

BU62382B14 RETRO2A4B14 FAB HRMS: C₂₃H₂₄N₂O₃SNa⁺ Predicted: 431.1405Found: 431.1405. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.69 (dd, J = 1.4, 7.7Hz, 1 H), 7.36 (d, J = 2.8 Hz, 1 H), 7.30-7.23 (m, 1 H), 7.14 (d, J =8.3 Hz, 1 H), 6.84 (d, J = 3.5 Hz, 1 H), 6.76-6.68 (m, 2 H), 6.65 (d, J= 3.5 Hz, 1 H), 6.59 (d, J = 2.5 Hz, 1 H), 6.52 (dd, J = 2.4, 8.5 Hz, 1H), 5.87 (d, J = 2.5 Hz, 1 H), 5.08 (d, J = 15.4 Hz, 1 H), 3.89 (d, J =15.4 Hz, 1 H), 3.79 (s, 3 H), 3.75 (s, 3 H), 2.69 (q, J = 7.5 Hz, 2 H),1.14 (t, J = 7.6 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.6, 160.0,158.1, 146.5, 146.0, 141.6, 133.4, 129.2, 127.6, 125.7, 122.7, 117.6,117.0, 115.0, 114.7, 104.7, 98.4, 66.5, 55.5, 55.2, 41.5, 22.7, 15.7.

BU62382B15 RETRO2A4B15 FAB HRMS: C₂₁H₁₉FN₂OSNa ⁺Predicted: 389.1100Found: 389.1109. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.69 (dd, J = 1.6, 7.7Hz, 1 H), 7.40 (d, J = 2.8 Hz, 1 H), 7.38-7.32 (m, 2 H), 7.31-7.25 (m, 1H), 7.19-7.12 (m, 2 H), 6.87 (d, J = 3.5 Hz, 1 H), 6.79-6.69 (m, 2 H),6.64 (td, J = 1.0, 3.5 Hz, 1 H), 5.94 (d, J = 2.5 Hz, 1 H), 5.16 (d, J =15.4 Hz, 1 H), 3.98 (d, J = 15.4 Hz, 1 H), 2.68 (dq, J = 0.9, 7.5 Hz, 2H), 1.13 (t, J = 7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d6) δ = 161.8,146.7, 146.2, 141.3, 133.8, 133.8, 133.5, 129.6, 129.5, 127.6, 127.4,126.1, 122.6, 117.7, 115.3, 115.1, 114.8, 114.8, 66.7, 46.3, 22.7, 15.7.

BU62382B16 RETRO2A4B16 FAB HRMS: C₂₁H₁₉FN₂OSNa⁺ Predicted: 389.1100Found: 389.1082. ¹H NMR (400 MHz, Acetone) δ = 7.70 (dd, J = 1.1, 8.0Hz, 1 H), 7.44 (d, J = 2.5 Hz, 1 H), 7.41-7.33 (m, 1 H), 7.29 (dt, J =1.6, 7.6 Hz, 1 H), 7.16 (d, J = 7.6 Hz, 1 H), 7.14- 7.05 (m, 2 H), 6.88(d, J = 3.3 Hz, 1 H), 6.78-6.71 (m, 2 H), 6.67-6.62 (m, 1 H), 6.00 (d, J= 2.5 Hz, 1 H), 5.16 (d, J = 15.7 Hz, 1 H), 4.06 (d, J = 15.7 Hz, 1 H),2.68 (q, J = 7.5 Hz, 2 H), 1.13 (t, J = 7.5 Hz, 3 H). ¹³C NMR (101 MHz,Acetone) δ = 163.5, 161.9, 161.0, 146.7, 146.3, 141.2, 140.8, 140.7,133.6, 130.4, 130.3, 127.6, 126.2, 123.4, 123.4, 122.6, 117.8, 114.9,114.7, 114.2, 114.0, 113.7, 67.0, 46.7, 22.7, 15.7.

BU62382B17 RETRO2A4B17 FAB HRMS: C₂₁H₁₉N₃O₃SNa⁺ Predicted: 416.1045Found: 416.1061. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.27-8.12 (m, 2 H), 7.69(dd, J = 1.5, 8.1 Hz, 1 H), 7.55 (d, J = 8.8 Hz, 2 H), 7.49 (d, J = 2.3Hz, 1 H), 7.36-7.25 (m, 1 H), 6.89 (d, J = 3.5 Hz, 1 H), 6.82-6.71 (m, 2H), 6.63 (td, J = 0.9, 3.5 Hz, 1 H), 6.07 (d, J = 2.5 Hz, 1 H), 5.15 (d,J = 16.2 Hz, 1 H), 4.29 (d, J = 16.2 Hz, 1 H), 2.67 (q, J = 7.4 Hz, 2H), 1.12 (t, J = 7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 162.1,146.9, 146.6, 146.4, 146.0, 141.1, 133.7, 128.4, 127.6, 126.4, 123.5,122.6, 117.8, 114.9, 114.6, 67.4, 47.0, 22.7, 15.8.

BU62382B18 RETRO2A4B18 FAB HRMS: C₂₁H₁₉N₃O₃SNa⁺ Predicted: 416.1045Found: 416.1038. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.17-8.04 (m, 2 H), 7.76(d, J = 7.8 Hz, 1 H), 7.74-7.67 (m, 1 H), 7.67-7.56 (m, 1 H), 7.48 (d, J= 2.3 Hz, 1 H), 7.30 (dt, J = 1.6, 7.6 Hz, 1 H), 6.88 (d, J = 3.5 Hz, 1H), 6.83-6.71 (m, 2 H), 6.66-6.58 (m, 1 H), 6.13 (d, J = 2.5 Hz, 1 H),5.07 (d, J = 15.4 Hz, 1 H), 4.39 (d, J = 15.7 Hz, 1 H), 2.65 (q, J = 7.5Hz, 2 H), 1.10 (t, J = 7.6 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ =162.2, 147.7, 146.8, 146.4, 141.3, 140.4, 134.3, 133.7, 129.9, 127.6,126.4, 122.6, 122.1, 117.8, 114.9, 114.6, 67.4, 46.9, 22.7, 15.7.

BU62382B19 RETRO2A4B19 FAB HRMS: C₂₂H₂₂N₂OSNa⁺ Predicted: 385.1351Found: 385.1342. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.71 (d, J = 7.8 Hz, 1H), 7.43-7.35 (m, 4 H), 7.34-7.28 (m, 1 H), 7.28-7.20 (m, 2 H), 6.81 (d,J = 3.3 Hz, 1 H), 6.74 (t, J = 7.5 Hz, 1 H), 6.65 (d, J = 8.1 Hz, 1 H),6.61 (d, J = 3.3 Hz, 1 H), 5.90 (q, J = 7.1 Hz, 1 H), 5.78 (d, J = 3.0Hz, 1 H), 2.66 (q, J = 7.7 Hz, 2 H), 1.36 (d, J = 7.3 Hz, 3 H), 1.12 (t,J = 7.6 Hz, 3 H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ = 161.6, 146.0,145.8, 143.6, 141.5, 133.3, 128.5, 127.8, 127.3, 126.8, 125.3, 122.5,117.8, 115.9, 115.0, 63.0, 51.0, 22.6, 17.5, 15.6.

BU62382B20 RETRO2A4B20 FAB HRMS: C₂₂H₂₂N₂OSNa⁺ Predicted: 385.1351Found: 385.1335. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.71 (dd, J = 1.5, 7.8Hz, 1 H), 7.42-7.35 (m, 4 H), 7.34- 7.28 (m, 1 H), 7.28-7.22 (m, 2 H),6.81 (d, J = 3.5 Hz, 1 H), 6.78-6.70 (m, 1 H), 6.65 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 3.5 Hz, 1 H), 5.90 (q, J = 7.2 Hz, 1 H), 5.78 (d, J =3.0 Hz, 1 H), 2.66 (q, J = 7.5 Hz, 2 H), 1.36 (d, J = 7.3 Hz, 3 H), 1.12(t, J = 7.6 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.5, 146.0,145.8, 143.6, 141.5, 133.3, 128.5, 127.8, 127.2, 126.8, 125.3, 122.5,117.8, 115.9, 115.0, 63.0, 51.0, 22.6, 17.5, 15.6

BU62382B21 RETRO2A4B21 FAB HRMS: C₂₂H₂₂N₂OSNa⁺ Predicted: 385.1351Found: 385.1369. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.65 (d, J = 7.8 Hz, 1H), 7.37 (s, 1 H), 7.33-7.25 (m, 3 H), 7.25-7.17 (m, 3 H), 6.92 (d, J =3.5 Hz, 1 H), 6.77-6.68 (m, 2 H), 6.68-6.62 (m, 1 H), 6.04 (d, J = 2.3Hz, 1 H), 4.09- 3.93 (m, 1 H), 3.14-3.01 (m, 1 H), 2.98- 2.84 (m, 1 H),2.81-2.71 (m, J = 5.1, 9.0, 9.0 Hz, 1 H), 2.68 (q, J = 7.3 Hz, 2 H),1.13 (t, J = 7.6 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.7, 146.7,146.2, 141.8, 139.1, 133.3, 128.6, 128.4, 127.4, 126.2, 125.9, 122.6,117.5, 114.9, 114.6, 67.0, 45.9, 33.6, 22.7, 15.7.

BU62382B15ox Retro2A4B15ox FAB HRMS: C₂₁H₁₇FN₂OSNa⁺ Predicted: 387.0943Found: 387.0938. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.14 (dd, J = 1.3, 8.3Hz, 1 H), 7.85 (dt, J = 1.4, 7.6 Hz, 1 H), 7.67 (d, J = 7.8 Hz, 1 H),7.59-7.50 (m, 1 H), 7.19-7.07 (m, 5 H), 6.83 (dd, J = 0.8, 3.8 Hz, 1 H),5.45 (s, 2 H), 2.81 (q, J = 7.5 Hz, 2 H), 1.23 (t, J = 7.5 Hz, 3 H). ¹³CNMR (101 MHz, DMSO-d₆) δ = 162.5, 161.8, 160.1, 152.0, 150.0, 147.0,135.0, 134.0, 133.1, 133.1, 129.6, 128.1, 128.0, 127.2, 126.6, 124.7,119.7, 115.8, 115.5, 48.2, 22.8, 15.8.

BU62382C1 Retro2A4B15C1 C₂₂H₂₁FN₂OSNa⁺ Predicted: 403.1256 Found:403.1266. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.80 (dd, J = 1.5, 7.6 Hz, 1 H),7.46-7.40 (m, 1 H), 7.40-7.33 (m, 2 H), 7.21-7.12 (m, 2 H), 6.93 (d, J =3.5 Hz, 1 H), 6.88 (dt, J = 1.0, 7.5 Hz, 1 H), 6.69 (d, J = 8.1 Hz, 1H), 6.65 (td, J = 1.0, 3.5 Hz, 1 H), 5.92 (s, 1 H), 5.13 (d, J = 15.4Hz, 1 H), 3.95 (d, J = 15.4 Hz, 1 H), 2.77 (s, 3 H), 2.68-2.60 (m, 2 H),1.11 (t, J = 7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.3, 147.0,146.4, 134.1, 133.5, 133.5, 129.6, 129.5, 127.8, 127.7, 122.6, 118.3,115.3, 115.1, 112.9, 73.3, 46.3, 34.9, 22.6, 15.5.

BU62382C2 Retro2A4B15C2 C₂₁H₁₈FN₂OSNa⁺ Predicted: 423.0710 Found:423.0718. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.71-7.65 (m, 2 H), 7.33 (dd, J= 5.7, 8.5 Hz, 2 H), 7.15 (t, J = 8.8 Hz, 2 H), 6.89 (d, J = 3.5 Hz, 1H), 6.79- 6.73 (m, 2 H), 6.68-6.63 (m, 1 H), 6.02 (d, J = 2.5 Hz, 1 H),5.12 (d, J = 15.4 Hz, 1 H), 3.99 (d, J = 15.4 Hz, 1 H), 2.69 (q, J = 7.5Hz, 2 H), 1.14 (t, J = 7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ =161.1, 160.2, 147.2, 147.0, 140.9, 138.1, 133.6, 133.6, 129.7, 129.6,129.5, 126.4, 122.8, 117.8, 115.3, 115.1, 113.9, 113.4, 66.7, 46.3,22.7, 15.7.

BU62382C3 Retro2A4B15C3 C₂₁H₁₈FN₂OSNa⁺ Predicted: 423.0710 Found:423.0725. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.63 (dd, J = 2.5, 5.3 Hz, 2 H),7.42-7.29 (m, 3 H), 7.20-7.12 (m, 2 H), 6.88 (d, J = 3.3 Hz, 1 H), 6.76(d, J = 8.8 Hz, 1 H), 6.67-6.63 (m, 1 H), 6.01 (d, J = 2.5 Hz, 1 H),5.13 (d, J = 15.2 Hz, 1 H), 4.01 (d, J = 15.2 Hz, 1 H), 2.68 (q, J = 7.4Hz, 2 H), 1.14 (t, J = 7.6 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ =160.7, 146.9, 145.0, 140.9, 133.4, 129.6, 129.6, 126.6, 126.3, 122.7,121.4, 116.9, 115.8, 115.3, 115.1, 66.6, 46.4, 22.7, 15.7.

BU62382C5 Retro2A4B15C5 C₂₂H₂₁FN₂OSNa⁺ Predicted: 403.1256 Found:403.1265. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.57 (d, J = 8.1 Hz, 1 H),7.45-7.32 (m, 2 H), 7.18 (t, J = 7.8 Hz, 3 H), 6.97 (d, J = 3.5 Hz, 1H), 6.81 (d, J = 3.3 Hz, 1 H), 6.71-6.65 (m, 1 H), 6.62 (d, J = 3.3 Hz,1 H), 5.90 (d, J = 3.8 Hz, 1 H), 5.27 (d, J = 15.9 Hz, 1 H), 4.04 (d, J= 14.9 Hz, 1 H), 2.67 (q, J = 7.6 Hz, 2 H), 2.07 (s, 3 H), 1.13 (t, J =7.6 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 162.1, 146.4, 142.0, 134.1,129.7, 129.6, 125.5, 125.5, 122.9, 122.8, 117.6, 115.4, 115.2, 66.1,46.5, 22.7, 16.8, 15.7.

BU62382C6 Retro2A4B15C6 C₂₂H₂₁FN₂OSNa⁺ Predicted: 403.1256 Found:403.1241. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.57 (d, J = 7.8 Hz, 1 H),7.37-7.29 (m, 3 H), 7.19-7.11 (m, 2 H), 6.85 (d, J = 3.5 Hz, 1 H), 6.63(td, J = 0.9, 3.4 Hz, 1 H), 6.56 (dd, J = 1.0, 8.1 Hz, 1 H), 6.51 (s, 1H), 5.90 (d, J = 2.5 Hz, 1 H), 5.15 (d, J = 15.2 Hz, 1 H), 3.95 (d, J =15.4 Hz, 1 H), 2.68 (q, J = 7.6 Hz, 2 H), 1.16- 1.11 (m, 3 H). ¹³C NMR(101 MHz, DMSO-d₆) δ = 162.1, 146.4, 143.9, 142.0, 134.1, 134.0, 133.9,129.6, 129.6, 125.5, 125.5, 122.9, 122.7, 117.5, 115.4, 115.2, 115.2,66.1, 46.5, 22.7, 16.8, 15.6.

BU62382C7 Retro2A4B15C7 C₂₂H₂₁FN₂OSNa⁺ Predicted: 403.1256 Found:403.1250. ¹H NMR (400 MHz, Acetone) δ = 7.50 (s, 1 H), 7.34 (dd, J =5.6, 8.1 Hz, 2 H), 7.23-7.07 (m, 4 H), 6.85 (d, J = 3.5 Hz, 1 H),6.71-6.54 (m, 2 H), 5.90 (d, J = 2.3 Hz, 1 H), 5.16 (d, J = 15.4 Hz, 1H), 3.97 (d, J = 15.4 Hz, 1 H), 2.67 (q, J = 7.4 Hz, 2 H), 2.21 (s, 3H), 1.13 (t, J = 7.3 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.9,146.6, 144.0, 141.4, 134.4, 129.6, 129.5, 127.5, 126.4, 126.1, 122.6,115.3, 115.1, 115.0, 114.8, 66.8, 46.3, 22.7, 20.1, 15.8.

BU62382C8 Retro2A4B15C8 C₂₂H₂₁FN₂OSNa⁺ Predicted: 403.1256 Found:403.1262. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.50 (s, 1 H), 7.34 (dd, J =5.6, 8.6 Hz, 2 H), 7.21 (d, J = 2.5 Hz, 1 H), 7.19-7.13 (m, 2 H), 7.11(dd, J = 2.0, 8.1 Hz, 1 H), 6.85 (d, J = 3.5 Hz, 1 H), 6.67- 6.60 (m, 2H), 5.89 (d, J = 2.5 Hz, 1 H), 5.16 (d, J = 15.2 Hz, 1 H), 3.97 (d, J =15.4 Hz, 1 H), 2.67 (q, J = 7.5 Hz, 2 H), 2.21 (s, 3 H), 1.13 (t, J =7.5 Hz, 3 H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 161.9, 146.6, 144.0, 141.4,134.4, 129.6, 129.5, 127.5, 126.4, 126.1, 122.6, 115.3, 115.1, 115.0,114.8, 66.8, 46.3, 22.7, 20.2, 15.8.

BU62382C9 Retro2A4B15C9 C₂₁H₁₈F₂N₂OSNa⁺ Predicted: 407.1006 Found:407.1025. ¹H NMR (600 MHz, DMSO-d₆) δ = 7.74 (dd, J = 6.6, 8.8 Hz, 1 H),7.66 (d, J = 2.6 Hz, 1 H), 7.37-7.30 (m, 2 H), 7.18-7.12 (m, 2 H), 6.89(d, J = 3.7 Hz, 1 H), 6.65 (td, J = 1.0, 3.6 Hz, 1 H), 6.54 (dt, J =2.4, 8.7 Hz, 1 H), 6.49 (dd, J = 2.2, 10.6 Hz, 1 H), 5.99 (d, J = 2.6Hz, 1 H), 5.13 (d, J = 15.4 Hz, 1 H), 3.98 (d, J = 15.4 Hz, 1 H), 2.69(q, J = 7.3 Hz, 2 H), 1.14 (t, J = 7.5 Hz, 3 H). ¹³C NMR (151 MHz,DMSO-d₆) δ = 165.4, 162.7, 161.6, 148.7, 147.4, 141.5, 134.1, 134.1,131.2, 131.1, 130.1, 130.0, 126.7, 123.2, 115.7, 115.6, 111.9, 105.7,105.6, 101.1, 100.9, 67.2, 46.7, 23.2, 16.2.

BU62382C10 Retro2A4B15C10 C₂₁H₁₈F₂N₂OSNa⁺ Predicted: 407.1006 Found:407.1018. ¹H NMR (400 MHz, DMSO-d₆) δ = 7.43-7.37 (m, 2 H), 7.34 (dd, J= 5.6, 8.3 Hz, 2 H), 7.23-7.12 (m, 3 H), 6.87 (d, J = 3.5 Hz, 1 H), 6.76(dd, J = 4.5, 8.8 Hz, 1 H), 6.66-6.62 (m, 1 H), 5.97 (d, J = 2.5 Hz, 1H), 5.12 (d, J = 15.4 Hz, 1 H), 4.02 (d, J = 15.2 Hz, 1 H), 2.68 (q, J =7.6 Hz, 2 H), 1.13 (t, J = 7.5 Hz, 3 H). ¹³C NMR (151 MHz, DMSO-d₆) δ =162.2, 161.0, 160.6, 155.8, 154.2, 146.7, 142.8, 140.9, 133.6, 133.6,129.6, 129.5, 126.2, 122.6, 121.1, 120.9, 116.7, 116.6, 115.5, 115.5,115.2, 115.1, 112.8, 112.6, 66.8, 46.4, 22.7, 15.6

FIG. 12 depicts the inhibitory properties of Retro-2^(cycl) analogsdepicted in Table 1. All compounds were tested against JCPyV at 25 uM in0.04% DMSO.

X-Ray Crystallography

A specimen of C₁₉H₁₆N₂OS, approximate dimensions 0.150 mm×0.250 mm×0.250mm, was used for the X-ray crystallographic analysis. The X-rayintensity data were measured on a Bruker Apex 1 diffractometer.

The integration of the data using a monoclinic unit cell yielded a totalof 5596 reflections to a maximum θ angle of 18.43° (1.12 Å resolution),of which 1181 were independent (average redundancy 4.738,completeness=98.5%, R_(int)=6.64%, R_(sig)=5.20%) and 899 (76.12%) weregreater than 2σ(F²). The final cell constants of a=6.575(6) Å,b=13.446(12) Å, c=18.616(17) Å, β=99.161(12)°, volume=1625.(3) Å³, arebased upon the refinement of the XYZ-centroids of reflections above 20σ(I). The calculated minimum and maximum transmission coefficients(based on crystal size) are 0.9506 and 0.9699.

The structure was solved and refined using the Bruker SHELXTL SoftwarePackage, using the space group P 2(1)/c, with Z=4 for the formula unit,C₁₉H₁₆N₂O S. The final anisotropic full-matrix least-squares refinementon F² with 227 variables converged at R1=5.01%, for the observed dataand wR2=13.47% for all data. The goodness-of-fit was 1.093. The largestpeak in the final difference electron density synthesis was 0.145 e⁻/Å³and the largest hole was −0.194 e⁻/Å³ with an RMS deviation of 0.039e⁻/Å³. On the basis of the final model, the calculated density was 1.310g/cm³ and F(000), 672 e⁻. Disorder in this crystal structure was notedfor the thiophene ring and the methine carbon atom of thedihydroquinazolinone ring to which it is attached. This disorder wasnicely modeled by a nearly 180° rotation about the C—C bond betweenC(14) and C(15) bond as well as an out of plane displacement of theC(14) relative to the remaining nine atoms of the dihydroquinazolinonering. Constraints were applied to the fragment C(14) through C(19) andS(1) in the initial stages of the refinement but were released in thefinal model. The final model refined to a nearly 2:1 ratio ofconformers. Only the major conformer is depicted in FIG. 8.

Other Embodiments

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1. A method of treating a viral infection, the method comprising administering to a subject suffering from or likely to suffer from a viral infection an effective amount of a compound of Formula (II):

or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof; wherein: X is O, S, or NH; each instance of R¹ is independently halo, —NO₂, —CN, —SCN, —OR^(A1), —SR^(A1), —N(R^(A1))₂, —C(═O)R^(A1), —OC(═O)R^(A1), —SC(═O)R^(A1), —NR^(A1)C(═O)R^(A1), —S(═O)₂R^(A1), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; each instance of R^(A1) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A1) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R¹ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R^(N) is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; each instance of R³ is independently halo, —NO₂, —CN, —SCN, —OR^(A3), —SR^(A3), —N(R^(A3))₂, —C(═O)R^(A3), —OC(═O)R^(A3), —SC(═O)R^(A3), —NR^(A3)C(═O)R^(A3), —S(═O)₂R^(A3), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, each instance of R^(A3) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A3) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R³ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R⁴ is hydrogen, substituted or unsubstituted alkyl, or an nitrogen protecting group; p is 0, 1, or 2; and m is 0, 1, 2, 3, or
 4. 2. (canceled)
 3. The method of claim 1, wherein the viral infection is an infection caused by human papillomavirus (HPV), human immunodeficiency virus (HIV), influenza virus, or polyomavirus. 4-9. (canceled)
 10. The method of claim 1, wherein X is S.
 11. The method of claim 1, wherein R¹ is halo or substituted or unsubstituted C₁₋₆ alkyl.
 12. The method of claim 11, wherein R¹ is methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), or n-hexyl (C₆).
 13. (canceled)
 14. The method of claim 1, wherein R^(N) is a group of formula:

wherein: L¹ is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, or substituted or unsubstituted alkynylene; each instance of R² is independently halo, —NO₂, —CN, —SCN, —OR^(A2), —SR^(A2), —N(R^(A2))₂, —C(═O)R^(A2), —OC(═O)R^(A2), —SC(═O)R^(A2), —NR^(A2)C(═O)R^(A2), —S(═O)₂R^(A2), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, each instance of R^(A2) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A2) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R² groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; and n is 0, 1, 2, 3, 4 or
 5. 15. The method of claim 14, wherein each instance of R² is independently halo, —NO₂, —OR^(A2).
 16. The method of claim 14, wherein L¹ is a bond.
 17. The method of claim 14, wherein L¹ is substituted or unsubstituted C₁alkylene, substituted or unsubstituted C₂alkylene, substituted or unsubstituted C₃alkylene.
 18. The method of claim 1, wherein m is 0 or
 1. 19. The method of claim 1, wherein each instance of R³ is independently halo or substituted or unsubstituted alkyl.
 20. The method of claim 1, wherein R⁴ is hydrogen.
 21. The method of claim 1, wherein the compound of Formula (II) is selected from the group consisting of:

and pharmaceutically acceptable salts, tautomers, prodrugs, and stereoisomers thereof.
 22. A method of treating a pathogenic condition associated with endosomal trafficking, the method comprising administering to a subject suffering from or likely to suffer from the condition an effective amount of a compound of Formula (II):

or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof; wherein: X is O, S, or NH; each instance of R¹ is independently halo, —NO₂, —CN, —SCN, —OR^(A1), —SR^(A1), —N(R^(A1))₂, —C(═O)R^(A1), —OC(═O)R^(A1), —SC(═O)R^(A1), —NR^(A1)C(═O)R^(A1), —S(═O)₂R^(A1), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; each instance of R^(A1) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A1) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R¹ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R^(N) is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; each instance of R³ is independently halo, —NO₂, —CN, —SCN, —OR^(A3), —SR^(A3), —N(R^(A3))₂, —C(═O)R^(A3), —OC(═O)R^(A3), —SC(═O)R^(A3), —NR^(A3)C(═O)R^(A3), —S(═O)₂R^(A3), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, each instance of R^(A3) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A3) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R³ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R⁴ is hydrogen, substituted or unsubstituted alkyl, or an nitrogen protecting group; p is 0, 1, or 2; and m is 0, 1, 2, 3, or
 4. 23. A method of treating an infection by a pathogen that secretes an AB₅ toxin, the method comprising administering to a subject suffering from or likely to suffer from the infection an effective amount of a compound of Formula (II):

or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof; wherein: X is O, S, or NH; each instance of R¹ is independently halo, —NO₂, —CN, —SCN, —OR^(A1), —SR^(A1), —N(R^(A1))₂, —C(═O)R^(A1), —OC(═O)R^(A1), —SC(═O)R^(A1), —NR^(A1)C(═O)R^(A1), —S(═O)₂R^(A1), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; each instance of R^(A1) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A1) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R¹ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R^(N) is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; each instance of R³ is independently halo, —NO₂, —CN, —SCN, —OR^(A3), —SR^(A3), —N(R^(A3))₂, —C(═O)R^(A3), —OC(═O)R^(A3), —SC(═O)R^(A3), —NR^(A3)C(═O)R^(A3), —S(═O)₂R^(A3), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, each instance of R^(A3) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A3) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R³ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R⁴ is hydrogen, substituted or unsubstituted alkyl, or an nitrogen protecting group; p is 0, 1, or 2; and m is 0, 1, 2, 3, or
 4. 24. The method of claim 23, wherein the pathogen secreting an AB₅ toxin is bacteria.
 25. (canceled)
 26. The method of claim 23, wherein the AB₅ toxin is selected from the group consisting of ricin, Shiga toxin, Shiga-like toxins, cholera toxin, heat-labile enterotoxin, pertussis toxin, and subtilase cytotoxin.
 27. A compound of Formula (II):

or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof; wherein: X is O, S, or NH; each instance of R¹ is independently halo, —NO₂, —CN, —SCN, —OR^(A1), —SR^(A1), —N(R^(A1))₂, —C(═O)R^(A1), —OC(═O)R^(A1), —SC(═O)R^(A1), —NR^(A1)C(═O)R^(A1), —S(═O)₂R^(A1), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; each instance of R^(A1) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A1) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R¹ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R^(N) is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; each instance of R³ is independently halo, —NO₂, —CN, —SCN, —OR^(A3), —SR^(A3), —N(R^(A3))₂, —C(═O)R^(A3), —OC(═O)R^(A3), —SC(═O)R^(A3), —NR^(A3)C(═O)R^(A3), —S(═O)₂R^(A3), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, each instance of R^(A3) is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted thiol, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom, or two R^(A3) groups are joined to form a substituted or unsubstituted heterocyclic or heteroaryl ring; or two R³ groups are joined to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclic, or substituted or unsubstituted heterocyclic ring; R⁴ is hydrogen, substituted or unsubstituted alkyl, or an nitrogen protecting group; p is 0, 1, or 2; and m is 0, 1, 2, 3, or 4; provided the compound is not:


28. The compound of claim 27, wherein the compound is:

or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.
 29. A pharmaceutical composition comprising a compound of claim 27, or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, and a pharmaceutically acceptable excipient. 